Methods and compositions for altering photophysical properties of fluorophores via proximal quenching

ABSTRACT

The invention is directed to fluorophore-containing compositions and configurations wherein proximity between the fluorophore and one or more protective agents (PAs) modifies the lifetime of fluorescent and/or dark states, their frequency of occurrence, and the total lifetime of fluorescence in order to appropriately modify the photophysical characteristics of the fluorophore. The invention is also directed to methods that utilize these compositions and configurations.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of copending application Ser. No.13/202,351 filed Sep. 20, 2011 which is a National Phase ofPCT/US2010/024824 filed Feb. 19, 2010 which claims benefit of priorityfrom U.S. Provisional Application 61/153,871 filed Feb. 19, 2009, all ofthe contents of which are incorporated herein by reference.

GOVERNMENT SUPPORT

This invention was made with government support under GM079238 awardedby the National Institutes of Health. The government has certain rightsin the invention.

FIELD OF THE INVENTION

The present invention relates generally to methods for identifyingcompounds with pharmaceutical properties, and more particularly, tomethods in which computer-generated structural information is includedin the method in order to identify compounds with improvedpharmaceutical properties.

BACKGROUND OF THE INVENTION

Fluorescence applications penetrate nearly every field of biologicalresearch and have more recently been proposed as a means for light-basedcomputational applications by virtue of a single fluorophore's observedpropensity to undergo a binary switch between fluorescent and darkstates. In vitro and in vivo fluorescence measurements, as well aswide-field, scanning confocal, and Total Internal ReflectionFluorescence Microscopy (TIRF) used for whole cell and single-moleculeimaging rely on high-quantum yield, stable fluorescent species such asthose shown in FIG. 3.

However, the utility of such fluorophores, including organic dyes,fluorescent proteins, as well as inorganic quantum dots andnanocrystals, is limited by their intrinsic photophysical propertiesthat lead to transient and/or permanent dark states. It is believed thatthese dark states arise via electronic transitions from the singletground and/or excited states to triplet dark states, as depicted by asimplified Jablonski diagram shown in FIG. 1. From triplet states,deleterious physical modifications or damage can occur to the dye.

For example, such processes tend to limit photon emission from thefluorophore including stochastic “blinking” events and irreversiblephotobleaching. Blinking and photobleaching phenomena occur in allfluorescence applications but are particularly pronounced in experimentsdemanding intense illumination, including confocal imaging of cells andsingle-molecule fluorescence methods.

In order to characterize the blinking and photobleaching behaviorsadequately, individual fluorophores must be tracked as a function oftime. Here, the application of modern single-molecule fluorescencemethods is ideal. Single, fluorescing molecules can be easily trackedusing total internal reflection (TIR) fluorescence methods where thefluorophore is spatially tethered near an optically-transparent surfaceand illuminated by a single-frequency laser light source, as shown, forexample, in FIG. 2. In such an experimental setting, blinking andphotobleaching appear as brief periods of fluorescence punctuated bylong-lived non-fluorescing states, as shown, for example in FIGS. 4A-4C.Although for some applications this switching behavior may bedetrimental, this characteristic may be harnessed (e.g., appropriatelyadjusted, modified, or even enhanced) for such applications assuper-resolution imaging, computational applications, and sensortechnologies.

Compounds such as Trolox, p-nitrobenzyl alcohol (NBA), β-mercaptoethanol(BME), mercaptoethylamine (MEA), n-propyl gallate,1,4-diazabicyclo[2.2.2]octane (DABCO), and cyclooctatetraene (COT) thatfavorably affect dark state and photobleaching lifetimes have come intoincreasingly widespread use as additives in solution-based experiments.However, the use of such protective agents in solution is limited bytheir solubility (Trolox, COT and NBA, in particular, are highlyinsoluble in aqueous solutions). Examples are shown in FIG. 5. Moreover,if beneficial outcomes are required for fluorescence imaging in cells,the protective agent's membrane permeability and potential toxicity mustalso be considered.

SUMMARY OF THE INVENTION

The present invention provides new fluorophore-containing compositionsand configurations wherein the fluorophore is placed in proximity to oneor more protective molecules (including triplet state quenchers (TSQs)that increase the rate of relaxation from the triplet dark state to theground state), which modifies the effective time spent in dark stateseither by reducing their frequency of occurrence or their lifetime inorder to appropriately modify the photophysical characteristics of thefluorophore. In particular, the proximity between the fluorophore andone or more protective agents can be adjusted in order to suitablyadjust the time spent in fluorescent and/or dark states. In particularembodiments, adjustments in proximity are made in order to improve thetotal number of photons emitted from a single fluorophore prior tophotobleaching and to increase or optimize the effective flux of photonsgenerated by a single fluorophore per unit time. Thus, the presentinvention establishes a general chemical strategy where dye proximity toone or more protective agents can be used to favorably “tune” dyephotophysics via the manipulation of dark state and fluorescent statelifetimes as schematized in FIG. 1.

Use of protective agents in solution as reported in the art is oftenlimited by protective agent solubility and/or toxicity in applicationsinvolving cells. Such limitations are bypassed by the present inventionwhere solubility considerations are circumnavigated by direct and/orindirect attachment of one or more protective agents to the dyemolecule. Accordingly, excess protective agent in solution is notrequired. In addition, the present invention effectively circumventsunwanted photophysical dye behavior in both bulk and single-moleculecontexts in the absence and presence of oxygen. In addition to improvingthe performance of dyes for fluorescence imaging experiments in vitro,this means of mitigating fluorophore photophysical processes can also beapplied to in vivo fluorescence and FRET imaging at both the bulk andsingle-molecule scale. One embodiment of single-molecule imaging whichdemands high-illumination intensity and long-lived fluorescence employsa total internal reflection configuration such as that shown in FIG. 2.The present invention can also be applied to molecular imaging whereincreased illumination intensities are demanded for applications such ashigh-spatial and -time resolution measurements; cellular imaging whereunwanted fluorophore photobleaching often limits the overall time andsignal-to-noise ratio of the measurement; super-resolution imaging,which demands robust dye lifetime and blinking kinetics PCR; sequencingand microarray applications that have ever-increasing demands onsensitivity; light-based computer applications where fluorophorephotobleaching determines the lifetime of the photoswitch; medicalimaging diagnostics based on fluorescence detection; as well asnanoparticles, such as quantum dots, impregnated with dye-protectiveagent conjugates.

In one embodiment, the invention is directed to a composition containinga fluorophore having attached thereto one or more protective agentmoieties. If two or more protective agent moieties are included, theymay be the same or different. The one or more protective agent moietiesare attached covalently or non-covalently to the fluorophore eitherdirectly or via a spacer molecule.

In another embodiment, the invention is directed to a compositioncontaining one or more fluorophores and one or more protective agentmolecules, wherein the fluorophores are in close proximity to theprotective agent such that the protective agent has an effect on thephotophysical properties of the fluorophore. In this embodiment, thefluorophore and protective agent are not linked directly or through alinker. For example, in one embodiment, the fluorophore and protectiveagent are separately conjugated to a biomolecule, such as a protein,DNA, RNA, RNA-protein, DNA-protein, or protein-protein complex. In aparticular embodiment, the protein is a fluorescent protein. In anotherembodiment, the fluorophore and protective agent are held proximal toeach other by having the fluorophore and protective agent incorporatedwithin different locations of a vesicle. In a further embodimentthereof, a biomolecule to be studied is encapsulated by the vesicle. Ina further embodiment thereof, the biomolecule is fluorophore-conjugated,and the protective agent is within the membrane of the vesicle or on aninner surface or outer surface of the membrane. In another embodiment,the fluorophore and protective agent are held proximal to each other byhaving the fluorophore and protective agent on the surface orpassivation shell of a nanoparticle. The nanoparticle can be, forexample, a quantum dot. In yet another embodiment, the fluorophore andprotective agent are held proximal to each other by having thefluorophore and protective agent attached to a surface of a bulk solid.

In another embodiment, the invention is directed to a method forperforming single-molecule Fluorescence Resonance Energy Transfer(FRET). The method includes measuring the FRET efficiency of a labeledbiomolecule containing a biomolecule attached to a donor fluorophore andacceptor fluorophore, wherein one or both of the fluorophores areattached covalently to one or more protective agent moieties, or one orboth of the fluorophores are not linked to a protective agent, eitherdirectly or via a linker, but held proximal to one or more protectiveagents. In a particular embodiment thereof, the fluorophore-labeledbiomolecule is encapsulated within a vesicle, and a protective agent iswithin a membrane of the vesicle or on an inner or outer surface of themembrane.

In yet another embodiment, the invention is directed to a method fortuning the photophysical properties of a fluorophore. The methodincludes analyzing the fluorescence properties of a consortium offluorophores held at proximal distances to one or more protectiveagents, and identifying new protective agents by this analysis, and/orfinding a fluorophore-protective agent composition with an optimal setof photophysical properties by this analysis.

In still another embodiment, the invention is directed to a method fordetecting a cellular process. The method includes: (i) administering tothe cell or animal a targeting probe that includes a biomoleculepossessing a targeting portion thereon, at least one fluorophore, and atleast one protective agent moiety, wherein the protective agent is inclose enough proximity to the fluorophore such that the protective agentenhances or otherwise optimizes the photophysical properties of thefluorophore, and ii) detecting the targeting probe in the cell.

In further embodiments of any of the compositions and methods describedabove, the fluorophore is a “red-shifted fluorophore”, i.e., byexhibiting an emission wavelength greater than 594 nm. Such fluorophoresare particularly suitable for use in FRET, and particularly, smFRETmethods.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Simplified Jablonski diagram describing competing kineticpathways that dictate the photophysical properties of fluorescentmolecular species. Shown here are the ground (S_(o)), singlet (S₁ andS_(N)), and triplet states (T₁ and T_(N)).

FIG. 2. Schematic of the Total Internal Reflection (TIR) imagingapproach. A single-fluorescently labeled species is tethered within theevanescent wave generated by TIR of one or more single-frequency lightsources. Fluorophore proximity to an optically-transparent surface isachieved by adhesion or through a specific, chemical strategy such as abiotin-streptavidin bridge as shown. In such a configuration,fluorescent properties can be interrogated and examined at high—spatialand—time resolution, comparable or better than those that can beachieved using confocal imaging methods.

FIG. 3. Single, fluorescent species of relevance to the presentinvention include organic fluorophores attached to biomolecules ofinterest (such as DNA or protein molecules as shown in the upperpanels), fluorescent proteins (such as mCherry shown in the lower leftpanel) and quantum dots (lower right panel).

FIGS. 4A-4C. Graphs showing blinking behavior typical of singlefluorescent dye molecules in 3 different experimental contexts:Cy5-labeled DNA oligonucleotides (top panel); a fluorescent proteinvariant mCherry (middle panel); quantum dot, an example of ananoparticle (bottom panel). In cellular conditions (which includesmolecular oxygen (O₂)) or under oxygen scavenging conditions (achievedby degassing or enzymatic means), fluorophores are fluorescent only afraction of the time (indicated by “Time On”, t_(on)). The remainder ofthe time is spent in one or more non-fluorescing states (indicated by“Time Off”, t_(off)) from which the return to fluorescence can often beslow even in the presence of “photoresurrecting” light sources.Temporary dark states are referred to in the art as “blinking”, eventsthought to result from intersystem crossing (ISC) to triplet states,while permanent dark states are “photobleached” states thought to resultfrom chemical damage to the fluorescing species.

FIG. 5. Exemplary “protective agents” which have been shown to mitigateunwanted photophysical processes. Shown are COT, Trolox and NBA andderivatives thereof.

FIGS. 6A-6D. Schemes for synthesizing Dye-Protective Agent conjugatesfrom commercially available fluorophores. Here, after linking an aminomodified protective agent (top panel NBA, bottom panel Trolox) to abis-NHS functionalized dye (Cy5), a reactive group is regenerated (NHSor maleimide in this example) for downstream coupling to a biomoleculeof interest.

FIG. 7. Structures of exemplary fluorophores containing reactivecoupling groups, and exemplary structures of fluorophore-protectiveagent (PA) compositions containing reactive coupling groups (NHS andmaleimide groups for example) for coupling with another molecule.Commercial fluorophores are often sold with a standard linker thatplaces the fluorophore at a set distance from the molecule to which itis attached. Fluorophore-PA molecules synthesized from commerciallyavailable agents are designed with the same linker in place, thusmaintaining the same specific distance from the molecule to which it isattached. Using synthetic strategies, the length of this linker can betailored according to experimental demand for either greater or lessmobility. Drawings provide a description of how new protectiveagent-containing dyes can be attached to biomolecules in a mannerconsistent with currently marketed dyes. Such dyes might be adventitiousbecause they retain the same distance between dye and biomolecule andtherefore allow for historical distance measurements to remain accurate.

FIGS. 8A-8B. Experimental data showing two examples of linking aprotective group to Cy3. Under direct excitation of Cy3, individualdwells in the fluorescent state were used to estimate time_(on)(t_(on)), the lifetimes in the fluorescent state. This distribution wasfit to an exponential decay process to demonstrate changes relative toCy3 without a PA. Cy3 fluorescence (t_(on)) is only marginally affectedby its direct linkage to NBA, while improved approximately 2.5-fold whendirectly linked to Trolox. Cy3- and Cy3-PA fluorescence behaviors wereinterrogated in the context of its linkage to DNA as schematized usingthe single-molecule TIR scheme show in FIG. 2. The specific dyemolecules examined are shown. These data demonstrate that distinctprotective agents operate either through distinct mechanisms or todifferent extents on the Cy3 fluorophore.

FIGS. 9A-9B. Experimental data taken as described in FIGS. 8A-8B hereshowing the lifetime of Cy5 fluorescence (t_(on)) is greatly improved(>10-fold) by its direct linkage to either NBA or Trolox. Cy5- andCy5-PA fluorescence behaviors were interrogated in the context of itslinkage to DNA as schematized using the single-molecule TIR scheme shownin FIG. 2. The specific dye molecules examined are shown. These datademonstrate that the same protective agents examined for the Cy3fluorophore operate either through distinct mechanisms or to differentextents on the Cy5 fluorophore.

FIGS. 10A-10B. Experimental data taken as described in FIGS. 8A-8Bshowing the lifetime of Cy5.5 fluorescence (t_(on)) is greatly improved(>20-fold) by its direct linkage to either NBA or Trolox. Cy5.5 andCy5.5-PA fluorescence behaviors were interrogated in the context of itslinkage to DNA as schematized using the single-molecule TIR scheme shownin FIG. 2. The specific dye molecules examined are shown. In yet anotherexample that distinct protective agents operate either through distinctmechanisms or to different extents on fluorophore species, Trolox andNBA are shown to improve the photophysical properties of the Cy5.5fluorophore more substantially than both Cy3 and Cy5.

FIGS. 11A-11D. Experimental data demonstrating improvements in theinformative FRET lifetime for the Cy3 and Cy5 pair. (Left panels)specific samples interrogated in which no protective agent is present,NBA is linked to Cy5, Trolox is linked Cy5 or a sample where distinctprotective agents are linked to each fluorophore: NBA is linked to Cy5and Trolox is linked Cy3. (Middle panels) Experimental single-moleculeFRET data showing examples of Cy3 and Cy5 fluorescence. Experimentaldata showing the population FRET behaviors observed where each of theconstructs containing fluorophore-PA conjugates has an extended FRETduration. In the sample where each fluorophore of the FRET pair islinked to a protective agent (bottom panels), the total lifetime of FRETis greatly improved over samples containing no protective agents(>20-fold) and significantly improved over samples where only theCy5-fluorophore is linked to a protective agent (>2-fold) (note the timeaxes on the bottom panels represent a much longer imaging period thanthe others shown). By increasing the FRET duration and minimizing darkstates, the information content of the experiment is substantiallyimproved. By extending the FRET duration, more robust structuralinformation can be obtained (as required for “Molecular EKG” imaging(Blanchard S C Curr Opin Struct. Biol Vol. 19(1) pg. 103-9 (2009))and/or greater confidence in the localization of single-fluorophoreswhen tracking blinking kinetics (as for super-resolution imaging).

FIGS. 12A-12C. Experimental data demonstrating another example of theimprovement in FRET lifetimes between a different FRET pair. In thiscase Cy3 and Cy5.5 are interrogated as described in FIGS. 11A-11D.Population histograms (right panels) reveal an increase in the lifetimeof fretting molecules when either of two protective agents are linked tothe Cy5.5 acceptor dye. Significantly, these data demonstrate anotherexample showing that protective agents linked to the acceptorfluorophore, Cy5.5 greatly extend the total period of FRET observationcompared to the case when no protective agents are present (>5-fold).

FIGS. 13A-13B. Experimental data showing the distance dependence ofprotective agent affects on fluorophore performance using Cy5-labeledDNA as a specific example. Protective agents NBA (left panel) or Trolox(right panel) were linked to 21-mer DNA oligonucleotides either directlythrough an NHS activating group on Cy5, or proximal to the fluorophorethrough a modified amine at the C6 position of internally designatedbases. Under direct excitation of Cy5, individual dwells in thefluorescent state were used to estimate t_(on), the lifetimes in thefluorescent state. This distribution was fit to a double exponentialdecay process and the time constants were divided by that of a no-PAcontrol to obtain the fold protection for each individual distance.These data demonstrate that the PA need not be directly attached to thefluorophore species in order to mediate control over the fluorophorephotophysics.

FIG. 14. Drawings depicting some exemplary specific, non-covalentgeometries and means for establishing close proximity betweenfluorescent molecules and one or more protective agents usingCy5-labeled DNA as a specific example (Upper left panel) proximity ofone or more protective agents to a fluorescing species by its attachmentto a nearby secondary molecule or chemical post (purple block). (Upperright panel) proximity of one or more protective agents to a fluorescingspecies by its attachment to a secondary molecule that non-covalentlyassociates with a lipid structure encapsulating the fluorescing species.(Lower left panel) proximity of one or more protective agents to afluorescing species by its attachment to a secondary molecule thatnon-covalently associates with the fluorescing species. (Lower rightpanel) proximity of one or more protective agents to amembrane-encapsulated fluorescing species achieved through theirattachment to, or embedding within, the inner or outer leaflet of alipid vesicle, liposome or nanosome.

FIG. 15. Drawings depicting some exemplary strategies for achievingproximity of one or more protective agents to a fluorescing species(Upper left panel) protective agents either chemically linked to naturalamino acid side chains located on the surface of a fluorescent proteinor by way of non-natural amino acid incorporation at the level ofprotein synthesis. (Upper right panel) one or more protective agentslinked to the outer shell layer(s) of a quantum dot by way ofimpregnation or covalent attachment. (Lower left panel) proximity of oneor more protective agents to a membrane-bound fluorescing speciesachieved through the attachment to, or embedded within, the inner orouter leaflet of a lipid vesicle, liposome or nanosome. (Lower rightpanel) proximity of one or more protective agents to a fluorescencespecies via embedding or impregnating the solid support to which thefluorescent species is located proximally.

FIG. 16. Drawings depicting some exemplary strategies for achievingproximity of more than one protective agent to a fluorescing speciesthat is attached to a target or substrate of interest as well asspecific examples of Cy5-PA linkages.

FIGS. 17A-17B. Experimental data demonstrating the utility of achievingproximity of more than one protective agent to a fluorescing speciesusing Cy5-labeled DNA as an example. As shown in FIGS. 9A-9B, theCy5-Trolox conjugate (orange) is greatly improved in lifetime over theno-PA Cy5 control (>10-fold). By adding a second protective agent(either NBA and Trolox) in proximity (within 30 Å) of the Cy5-Troloxfluorophore, an additional improvement in fluorophore lifetime (t_(on))is observed. In the case of a Trolox (purple), the additionalimprovement is >1.3-fold. In the case of NBA (green) the additionalimprovement is >2-fold. Thus, the total improvement achievedwas >20-fold over the parent compound. Drawings depicting otherexemplary strategies for achieving proximity of more than one protectiveagent to a fluorescing species.

FIG. 18. Schematic representation of the tuning of fluorescencebehavior. The relevant parameters are the lifetimes in fluorescingstates (Time on, t_(on)) and non-fluorescing states (Time off, t_(off))and the fluorescence intensities. The present invention describes howone can change the intrinsic fluorescent properties of a fluorescingspecies by controlling fluorophore proximity to one or more protectiveagents. Such strategies can be used to achieve long-lasting fluorescingstates and shorter dark states or shorter-lived fluorescent and darkstates depending on the specific PAs employed and their proximity to thefluorescent species. Similar strategies may also be used to control thequantum yield (brightness) of a fluorophore under a given illuminationintensity.

FIG. 19. Experimental data showing that under conditions similar tothose found in the cell (buffers saturated (1-2 mM) with molecularoxygen (O₂)) fluorophore-PA proximity extends the lifetime offluorescence (t_(on)). (Upper panel) Specific, exemplary fluorophore-PAconjugates under direct excitation (namely Cy5-trolox and Cy5-NBA)(Lower panel) Quantum dots bathed in a solution of 1 mM Trolox, COT andNBA. In each case the distribution of fluorescence lifetimes (ton) werefit to an exponential process.

FIG. 20. Drawing encompassing various embodiments of the invention inwhich a protective agent is connected to a fluorophore via a ‘linker’,[XY_(n)]_(m).

FIG. 21. Drawings showing some exemplary Cy3-NBA and Cy3-Troloxcompositions of the invention.

FIG. 22. Drawings showing some exemplary NHS-Cy5.5-NBA andNHS-Cy5.5-Trolox compositions of the invention.

FIGS. 23A-23B. Drawings showing a commercial mono-reactive NHS-Cy5composition, bis-reactive NHS-Cy5 composition, mono-reactive Mal-Cy5composition, and bis-reactive Mal-Cy5 composition.

FIGS. 24A-24B. Drawings showing some exemplary NHS-Cy-NBA,NHS-Cy5-Trolox, Mal-Cy5-NBA, and Mal-Cy5-Trolox compositions of theinvention.

FIGS. 25A-25B. Drawings showing the attachment of one or more protectiveagents to membrane structures through reactive elements either naturallyoccurring or introduced into phospholipid head groups or detergents orsterol compounds such as cholesterol that intercalate/impregnatephospholipid bilayers and detergent micelles.

FIGS. 26A-26B. Drawing showing exemplary chemical strategies for theintroduction of protective agents onto the exterior of quantum dotcompounds. In the cases shown, the protective agent bound can be of asingle type, or of multiple types.

FIG. 27. Drawing showing an exemplary fluorophore-protecting agentconjugate covalently bound to a piece of RNA. Exemplary chemicalstructure of fluorophore-protective agent conjugate covalently attachedto an RNA molecule through a specific reactive moiety present in thefluorophore.

FIG. 28. Strategy whereby a fluorophore-protective agent conjugate islinked to a reporter fragment via a reactive sulhydryl moiety in closeproximity to a quenching agent-cleavage fragment domain. The utility ofsuch probes is exemplified in Barnett, E. A., et al., Proc. Nat. Acad.Sci. 2009, 106, 9391-9396. The specific example shown is a peptideconstruct, but may also be applicable to DNA and/or RNA. Cleavage of thefluorophore-PA conjugate from the cleavage domain liberates thefluorophore-PA conjugate from proximity to the quencher, therebyproducing a detectable FRET signal. By modifying the photophysicalproperties of the liberated fluorophore, the present invention improvesone's ability to detect the cleavage reaction via fluorescence.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, a “fluorophore” (or “fluorescing species”) refers to anyspecies possessing a fluorescent property when appropriately stimulated.The stimulation that elicits fluorescence is typically illumination;however, other types of stimulation (e.g., collisional) are alsoconsidered herein.

In one embodiment, the fluorophore is an organic fluorophore. Indifferent embodiments, the organic fluorophore can include, for example,a charged (i.e., ionic) molecule (e.g., sulfonate or ammonium groups),uncharged (i.e., neutral) molecule, saturated molecule, unsaturatedmolecule, cyclic molecule, bicyclic molecule, tricyclic molecule,polycyclic molecule, acyclic molecule, aromatic molecule, and/orheterocyclic molecule (i.e., by being ring-substituted by one or moreheteroatoms selected from, for example, nitrogen, oxygen and sulfur). Inthe particular case of unsaturated fluorophores, the fluorophorecontains one, two, three, or more carbon-carbon and/or carbon-nitrogendouble and/or triple bonds. In a particular embodiment, the fluorophorecontains at least two (e.g., two, three, four, five, or more) conjugateddouble bonds aside from any aromatic group that may be in thefluorophore. In other embodiments, the fluorophore is a fused polycyclicaromatic hydrocarbon (PAH) containing at least two, three, four, five,or six rings (e.g., naphthalene, pyrene, anthracene, chrysene,triphenylene, tetracene, azulene, and phenanthrene) wherein the PAH canbe optionally ring-substituted or derivatized by one, two, three or moreheteroatoms or heteroatom-containing groups.

In other embodiments, the organic fluorophore is a xanthene derivative(e.g., fluorescein, rhodamine, Oregon green, eosin, and Texas Red),cyanine or its derivatives or subclasses (e.g., streptocyanines,hemicyanines, closed chain cyanines, phycocyanins, allophycocyanins,indocarbocyanines, oxacarbocyanines, thiacarbocyanines, merocyanins, andphthalocyanines), naphthalene derivatives (e.g., dansyl and prodanderivatives), coumarin and its derivatives, oxadiazole and itsderivatives (e.g., pyridyloxazoles, nitrobenzoxadiazoles, andbenzoxadiazoles), pyrene and its derivatives, oxazine and itsderivatives (e.g., Nile Red, Nile Blue, and cresyl violet), acridinederivatives (e.g., proflavin, acridine orange, and acridine yellow),arylmethine derivatives (e.g., auramine, crystal violet, and malachitegreen), and tetrapyrrole derivatives (e.g., porphyrins and bilirubins).Some particular families of dyes considered herein are the Cy® family ofdyes (e.g., Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, and Cy7), the Alexa®family of dyes (e.g., Alexa Fluor 350, 405, 430, 488, 500, 514, 532,546, 555, 568, 594, 610, 633, 647, 660, 680, 700, 750, and 790), theATTO® family of dyes (e.g., ATTO 390, 425, 465, 488, 495, 520, 532, 550,565, 590, 594, 601, 615, 619, 629, 635, 645, 663, 680, 700, 729, and740), and the Dy® family of dyes (e.g., DY 530, 547, 548, 549, 550, 554,556, 560, 590, 610, 615, 630, 631, 631, 632, 633, 634, 635, 636, 647,648, 649, 650, 651, 652, 675, 676, 677, 680, 681, 682, 700, 701, 730,731, 732, 734, 750, 751, 752, 776, 780, 781, 782, and 831). The ATTOdyes, in particular, can have several structural motifs, including,coumarin-based, rhodamine-based, carbopyronin-based, and oxazine-basedstructural motifs.

In a particular embodiment, the fluorophore is a streptocyanine (openchain cyanine) having the general structure:

wherein n in formula (1) above can be, for example, precisely, at least,or no more than 0, 1, 2, 3, 4, 5, 6, 7, 8, or within a range therein.Other structures related to or derived from formula (1) are alsoconsidered herein, as amply described in Guieu, V., et al., Eur. J. Org.Chem., 2007, 804-810, which is incorporated herein by reference in itsentirety. A particular streptocyanine molecule considered herein has thefollowing structure:

In another particular embodiment, the fluorophore is a hemicyaninehaving the general structure:

wherein n in formula (2) is as defined above. Other structures relatedto or derived from formula (2) are also considered herein, as amplydescribed in Stathatos, E., et al. Chem. Mater., 2001, 13, 3888-3892,and Yao, Q.-H., et al. J. Mater. Chem., 2003, 13, 1048-1053, which areincorporated herein by reference in their entirety. Some particularstreptocyanine molecules considered herein have the followingstructures:

In another particular embodiment, the fluorophore is a closed cyaninehaving the general structure:

wherein n in formula (3) is as defined above. Some particularstreptocyanine molecules considered herein have the followingstructures:

In another particular embodiment, the fluorophore is a coumarin-basedmolecule having the general structure:

Other structures related to or derived from formula (4) are alsoconsidered herein, as amply described in Schiedel, M.-S., et al. J.Organomet. Chem. 2002, 653, 200-208, which is incorporated herein byreference in its entirety. Some particular coumarin-based moleculesconsidered herein have the following structures:

In another particular embodiment, the fluorophore is a rhodamine-basedmolecule having the general structure:

Other structures related to or derived from formula (5) are alsoconsidered herein, as amply described in Beija, M., et al. Chem. Soc.Rev. 2009, 38, 2410-2433, which is incorporated herein by reference inits entirety. Some particular rhodamine-based molecules consideredherein have the following structures:

In another particular embodiment, the fluorophore is acarbopyronin-based molecule having the general structure:

Other structures related to or derived from formula (6) are alsoconsidered herin, as amply described in Arden-Jacob, J., et al.Spectrochim. Acta 2001, 57, 2271-2283, which is incorporated herein byreference in its entirety. Some particular carbopyronin-based moleculesconsidered herein have the following structures:

In another particular embodiment, the fluorophore is a oxazine-basedmolecule having the general structure:

Other structures related to or derived from formula (7) are alsoconsidered herein, as amply described by Van Thien, T. in Chemistry andApplications of Leuco Dyes 1997, 67-95, which is incorporated herein byreference in its entirety. A particular oxazine-based moleculeconsidered herein has the following structure:

In another particular embodiment, the fluorophore consists of at leasttwo (e.g., two, three, four, five, or more) conjugated double bondsaside from any aromatic group that may be in the fluorophore. Suchcompounds may not neccessarily be classified as belonging to aparticular group. Some particular compounds of this nature consideredherein have the following structures:

The fluorophores considered herein can absorb and emit light of anywavelengths. However, in different embodiments, it may be desired toselect a fluorophore with particular absorption and emissioncharacteristics. For example, in different embodiments, the fluorophorepreferably absorbs at nanometer (nm) wavelengths of 200, 210, 220, 230,240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370,380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510,520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650,660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, or800 nm, or within a range bounded by any two of the foregoing values. Indifferent embodiments, the fluorophore preferably emits at any of theforegoing wavelengths, or within a range bounded by any two of theforegoing values, wherein it is understood that a fluorophore generallyemits at a higher wavelength than the absorbed wavelength. The impingingelectromagnetic radiation (i.e., which is absorbed by the fluorophore)can be in a dispersed form, or alternatively, in a focused form, such asa laser. Moreover, the absorbed or emitted radiation can be in the formof, for example, far infrared, infrared, far red, visible,near-ultraviolet, or ultraviolet. When two or more fluorophores are used(e.g., attached to a biomolecule, as in FRET and smFRET methods), one ofthe fluorophores functions as a donor fluorophore and the otherfunctions as an acceptor fluorophore. In some embodiments, it ispreferred for a protective agent to bind to or be in close proximitywith either the acceptor fluorophore or the donor fluorophore, but notboth.

In particular embodiments, a “red-shifted fluorophore” is preferred. Thered-shifted fluorophore is characterized by exhibiting an emissionwavelength greater than 594 nm. Such fluorophores are particularlyuseful in FRET and small molecule FRET (i.e., smFRET) methods.

In another embodiment, the fluorophore has an inorganic composition. Forexample, the fluorophore can include a fluorescent transition metal orrare earth (e.g., lanthanide) metal species or particle (e.g.,nanoparticle or microparticle). The transition metal or rare earth metalspecies can be, for example, a metal-ligand complex. The ligand can beany suitable ligand, such as, for example, acetylacetonate, a Schiffbase (e.g., salen), amine, phosphine, thiol, phenanthroline, bipyridine,or phenolate-based ligand. By “transition metal” is meant any of themetals in Groups IB to VIIIB of the Periodic Table. By “rare earthmetal” is meant any of the lanthanides and actinides with atomic numbersof, respectively, 57-71 and 90-103. Some particular rare earth metalsconsidered herein include lanthanum, cerium, praseodymium, neodymium,promethium, samarium, europium, gadolinium, terbium, dysprosium,holmium, erbium, thulium, ytterbium, lutetium, and hafnium, andcombinations thereof.

In one embodiment, a fluorescent nanoparticle contains a non-fluorescentmatrix (e.g., a polymer, ceramic, or metal) into which is included anorganic or inorganic fluorescent species (i.e., within or on the surfaceof the nanoparticle). In particular embodiments, the particle includesan organic polymer (e.g., polystyrene) or inorganic polymer (e.g.,silica or siloxane-based). In another embodiment, the particle includesa metal, e.g., nanoparticles based on copper, silver, gold, palladium,or platinum, or a combination thereof. In another embodiment, thefluorescent particle has a semiconductor (i.e., quantum dot)composition. The quantum dot particle typically includes a sulfide,selenide, telluride, nitride, phosphide, arsenide, and/or antimonide ofa Group IB element (e.g., Cu or Ag), Group II element (e.g., Zn or Cd),or Group III element (e.g., B, Al, Ga, or In), or combination of theseelements. Furthermore, the quantum dot can be essentially homogeneous instructure, or alternatively, layered (e.g., a core-shell quantum dot).The core and shell of such a quantum dot can, independently, be composedof any of the semiconductor compositions described above (e.g.,“core:shell” compositions of the type ZnS:CdSe, ZnSe:CdSe, ZnS:CdS,ZnSe:CdS, CdS:ZnSe, CdSe:ZnSe, CdS:ZnS, or CdSe:ZnS). In someembodiments, the quantum dot further includes a dopant fluorescentspecies, such as any of the rare earth metals or organic fluorescentspecies described above, either by being incorporated into the core orshell of the quantum dot or by being adsorbed or linked to the surfaceor passivation shell of the quantum dot.

In different embodiments, the particle (particularly, a quantum dot)possesses a size (i.e., diameter) of, for example, 1, 2, 5, 10, 15, 20,30, 40, 50, 60, 70, 80, 90, 100, 110, or 120 nm, or a size within arange bounded by any two of the foregoing values. A metal particle thatis not a quantum dot may be any of the foregoing sizes, as well assignificantly larger sizes, e.g., about, at least, or no more than 150,200, 250, 300, 350, 400, or 500 nm or a size within a range bounded byany two of the foregoing values. A polymer particle can be any of theforegoing sizes, as well as significantly larger sizes, e.g., about, atleast, or no more than 600, 700, 800, 900, 1000, 2000, 3000, 4000, or5000 nm, or a size within a range bounded by any two of the foregoingvalues.

Any of the fluorophores considered herein (and particularly, the organicfluorophores) can include functional groups that allow them to be linkedto another molecule or conjugated to a biomolecule. For example, thefluorophore can include one, two, or more amino-reactive,thiol-reactive, aldehyde-reactive, or ketone-reactive groups. Someexamples of amino-reactive groups include activated ester groups (e.g.,N-succinimidyl ester (e.g., N-hydroxysuccinimidyl, i.e., NHS),carbodiimide ester groups (e.g., EDC), tetrafluorophenyl esters,dichlorophenol esters, and sulfo-derivatives thereof, and combinationsthereof), isothiocyanate, sulfonylchloride, dichlorotriazines, arylhalides, and acyl azides. Some examples of thiol reactive groups includemaleimido groups, haloacetamide (e.g., iodoacetamide) groups, disulfidegroups, thiosulfate, and acryloyl groups. Some examples ofaldehyde-reactive and ketone-reactive groups include hydrazide,semicarbazide, carbohydrazide, and hydroxylamine groups. The functionalgroup can also be a reactive probe, i.e., a chemical moiety thatselectively targets (i.e., binds to and/or reacts with) anothermolecule. In a preferred example, the reaction is specific for the tworeacting species such that reaction of the probe with another functionalgroup on the molecule of interest is not possible (e.g., reactionsreviewed in Prescher, J. A., et al., Nat. Chem. Biol. 2005, 1, 13-21,which is incorporated herein by reference in its entirety). For example,a biotin molecule may be included in the fluorophore to specificallybind the fluorophore to a streptavidin conjugate; or an antibody orfragment thereof may be included in the fluorophore to specifically bindthe fluorophore to a molecule bearing an epitope reactive with theantibody; or a peptide, oligopeptide, or lectin may be included in thefluorophore to specifically bind or react the fluorophore with anotherbiomolecule; or a nucleic acid, nucleoside, nucleotide, oligonucleotide,or DNA or RNA strand or vector may be included in the fluorophore tospecifically bind the fluorophore to a complimentary strand.

As used herein, a “protective agent” (or “quencher” or “triplet statequencher” or “fluorescence modifier”, in particular embodiments) is amolecule or a moiety (i.e., group) that has the ability to alter thephotophysical properties of a fluorophore, particularly by altering thelight state-dark state (i.e., singlet-triplet) occupancy distribution orrelaxation pathway of excited and relaxing electrons. The ability of amolecule to function as a protective agent is often evidenced by itsability to alter the blinking and/or photobleaching characteristics of afluorophore.

In a particular embodiment, the protective agent is a chromanol-derivedmolecule within the following generic structural formula:

In formula (9) above, R₁, R₂, R₃, R₄ and R₅ can independently beselected from a hydrogen atom or hydrocarbon group. The hydrocarbongroup can be saturated or unsaturated, and/or straight-chained orbranched, and/or cyclic or acyclic, and can include, for example, one,two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, ormore carbon atoms (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl,isobutyl, t-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, anddodecyl groups). An unsaturated hydrocarbon group can contain at leastone, two, three, or more carbon-carbon or carbon-nitrogen double and/ortriplet bonds. In one embodiment, the hydrocarbon group is composedsolely of carbon and hydrogen. In another embodiment, the hydrocarbongroup includes one, two, three, or more heteroatoms, such as oxygen,nitrogen, sulfur, and/or halogen (e.g., fluoro, chloro, bromo, iodo)atoms. Since the protective agent molecules described herein are oftendirectly or indirectly covalently linked to a fluorophore and/orbiomolecule, a surface (i.e., of a bulk solid), or other molecule ofinterest, any one or more of the groups R₁, R₂, R₃, R₄ and R₅ can alsorepresent a bond to another molecule or surface, or alternatively, alinking group with a bond to another molecule or a surface. Often, thegroup R₄ can be taken as a bond to an atom of a molecule, macromolecule,or surface.

In particular embodiments of formula (9), R₁, R₂, and R₃ are selectedfrom methyl and hydrogen atoms (i.e., all methyl groups, or two methylgroups and one hydrogen atom, or one methyl group and two hydrogenatoms, or all hydrogen atoms). In another particular embodiment, R₄ is amethyl or carboxylic acid group, or a methylene, ethylene, carbonyl,ester, or amido linking group. In other embodiments, R₄ and R₅ areindependently selected from methyl, ethyl, vinyl, allyl, n-propyl,n-butyl, isobutyl, t-butyl, and/or hydrogen (H) groups. In particularembodiments, R₄ and R₅ are both methyl groups, both hydrogen atoms, orone is methyl and the other hydrogen. In other particular embodiments,R₅ is a long chain hydrocarbon group (e.g., of at least 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, or 16 carbon atoms).

In more specific embodiments, R₅ is an unsaturated group that results informula (9) being a tocopherol, or tocotrienol, or derivative thereof;for example, wherein R₅ has the structure:

In a particular embodiment, the protective agent is Trolox, which hasthe following formula:

Formula (11) is also meant to include any derivative of Trolox thatretains protective agent abilities. The Trolox derivative can be, forexample, a result of replacing the carboxy OH group by a hydrocarbongroup (e.g., any of the hydrocarbon groups described above), an ethergroup (i.e., —OR group, where R is a hydrocarbon group orester-activating group), an amino group (i.e., —NR₆R₇ group, where R₆and R₇ are selected from hydrogen atoms and/or hydrocarbon groups),β-keto group, sulfonamide group, phosphate group, or chloro group. TheTrolox derivative can also be, for example, a result of replacing thephenolic OH group or a hydrogen atom with any of the foregoing groups.Formula (11) is also meant to include a Trolox or Trolox derivativegroup bound to an atom of another molecule (wherein the molecule may be,for example, a fluorophore, a linker, biomolecule, macromolecule, orsurface). The Trolox group results by replacement of one or morehydrogen atoms of Trolox or derivative thereof with a bond (or with alinker having a bond) to another molecule or surface. The hydrogen atombeing replaced with a bond can be, for example, from a C—H group,hydroxyl group, amino group, or other group. Alternatively, the Troloxgroup results by replacement of one or more hydroxyl groups, methylgroups, or the carboxy group with a bond (or with a linker having abond) to another molecule or surface.

In another particular embodiment, the protective agent is anitro-substituted aromatic molecule or group in which the aromaticmolecule or group can be cyclic (i.e., contain a single ring) orpolycyclic (i.e., two, three, four, or more rings either linked or fusedwith each other). Typically, the molecule contains one or two nitrogroups per molecule or ring. Some examples of nitro-substituted aromaticmolecules include nitrobenzene, the nitrotoluenes, picric acid, thenitronaphthalenes, nitrobiphenyls, and nitro derivatives of any of thepolycyclic aromatic hydrocarbons described above, and derivativesthereof (e.g., inclusion of one or more hydroxyl, hydroxyalkyl, ether,or carboxamido groups). Some examples of nitro-substituted aromaticgroups include those resulting from removal of a hydrogen atom from anyof the foregoing exemplary molecules and replacing this with a bond toan atom of another molecule, a surface, or a macromolecule. For example,removal of a methyl hydrogen atom from a p-nitrotoluene molecule resultsin a p-nitrobenzyl group. In particular embodiments, thenitro-substituted aromatic molecule is o-, m-, or p-nitrobenzyl alcohol(NBA), 2,6-dinitrobenzyl alcohol, 3,4-dinitrobenzyl alcohol, ahalo-substituted nitrobenzyl alcohol, or chloroamphenicol. In otherembodiments, the nitro-substituted aromatic molecule is o-, m-, orp-nitrobenzyl amine, or derivatives thereof.

In a particular embodiment, the protective agent is a nitro-substitutedaromatic molecule or group within the following generic structuralformula:

In formula (12) above, R₁, R₂, R₃, R₄, R₅, R₆, R₇, and R₈ canindependently be selected from a hydrogen atom or hydrocarbon group. Thehydrocarbon group can be saturated or unsaturated, and/orstraight-chained or branched, and/or cyclic or acyclic, and can include,for example, one, two, three, four, five, six, seven, eight, nine, ten,eleven, twelve, or more carbon atoms (e.g., methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, t-butyl, pentyl, hexyl, heptyl, octyl,nonyl, decyl, and dodecyl groups). An unsaturated hydrocarbon group cancontain at least one, two, three, or more carbon-carbon orcarbon-nitrogen double and/or triplet bonds. In one embodiment, thehydrocarbon group is composed solely of carbon and hydrogen. In anotherembodiment, the hydrocarbon group includes one, two, three, or moreheteroatoms, such as oxygen, nitrogen, sulfur, and/or halogen (e.g.,fluoro, chloro, bromo, iodo) atoms. Since the protective agent moleculesdescribed herein are often directly or indirectly covalently linked to afluorophore and/or biomolecule, a surface (i.e., of a bulk solid), orother molecule of interest, any one or more of the groups R₁, R₂, R₃,R₄, R₅, R₆, R₇, and R₈ can also represent a bond to another molecule orsurface, or alternatively, a linking group with a bond to anothermolecule or a surface. Often, the group R₁ can be taken as a bond to anatom of a molecule, macromolecule, or surface.

In particular embodiments of formula (12), R₁, R₂, and R₃ are selectedfrom nitrogen, oxygen, carbon, and hydrogen atoms with the proviso thatat least one of them is either nitrogen or oxygen.

In more specific embodiments, R₁ is an unsaturated group that results informula (12) being a nitrobenzyl alcohol, or derivative thereof; forexample, wherein R₁ has the structure:

In more specific embodiments, R₁ is an unsaturated group that results informula (12) being a nitrobenzyl amine, or derivative thereof; forexample, wherein R₁ has the structure:

In more specific embodiments, R₁ is an unsaturated group that results informula (12) being a nitrobenzyl ethane-1,2-diamine, or derivativethereof; for example, wherein R₁ has the structure:

In more specific embodiments, R₁ is an unsaturated group that results informula (12) being a 2-(nitrobenzylamino) ethanol, or derivativethereof; for example, wherein R₁ has the structure:

In a particular embodiment, the protective agent of formula (12) isN¹-(4-nitrobenzyl)ethane-1,2-diamine, which has the following formula:

Formula (17) is also meant to include any derivative ofN¹-(4-nitrobenzyl)ethane-1,2-diamine that retains protective agentabilities. Such a derivative can be, for example, a result of replacingeither one or both of the amines with another heteroatom (i.e., oxygen,sulfur, or phosphorous), a sulfonamide group, or phosphate group. TheN¹-(4-nitrobenzyl)ethane-1,2-diamine derivative can also be, forexample, a result of replacing a hydrogen atom with a nitrogen atom orany of the foregoing groups. Formula (17) is also meant to includeN¹-(4-nitrobenzyl)ethane-1,2-diamine orN¹-(4-nitrobenzyl)ethane-1,2-diamine derivative bound to an atom ofanother molecule (wherein the molecule may be, for example, afluorophore, a linker, biomolecule, macromolecule, or surface). TheN¹-(4-nitrobenzyl)ethane-1,2-diamine derivative results by replacementof one or more hydrogen atoms of N¹-(4-nitrobenzyl)ethane-1,2-diamine orderivative thereof with a bond (or with a linker having a bond) toanother molecule or surface. The hydrogen atom being replaced with abond can be, for example, from a C—H group, hydroxyl group, amino group,or other group.

In a particular embodiment, the protective agent is Chloramphenicol,which has the following structure:

Formula (18) is also meant to include any derivative of Chloramphenicolthat retains protective agent abilities. The Chloramphenicol derivativecan be, for example, a result of replacing one or both OH groups by ahydrocarbon group (e.g., any of the hydrocarbon groups described above),an ether group (i.e., —OR group, where R is a hydrocarbon group orester-activating group), an amino group (i.e., —NR₆R₇ group, where R₆and R₇ are selected from hydrogen atoms and/or hydrocarbon groups),β-keto group, sulfonamide group, phosphate group, or chloro group. TheChloramphenicol derivative can also be, for example, a result ofreplacing the amide moiety with any of the foregoing groups. Formula(18) is also meant to include a Chloramphenicol or Chloramphenicolderivative group bound to an atom of another molecule (wherein themolecule may be, for example, a fluorophore, a linker, biomolecule,macromolecule, or surface). The Chloramphenicol group results byreplacement of one or more hydrogen atoms of Chloramphenicol orderivative thereof with a bond (or with a linker having a bond) toanother molecule or surface. The hydrogen atom being replaced with abond can be, for example, from a C—H group, hydroxyl group, amino group,or other group. Alternatively, the Chloramphenicol group results byreplacement of one or more hydroxyl groups, chloro groups,dichloromethyl group, or the amide group with a bond (or with a linkerhaving a bond) to another molecule or surface.

In another embodiment, the protective agent is a conjugated polyenemolecule or group. The conjugated polyene considered herein can be, forexample, straight-chained or branched, and either cyclic or acyclic. Indifferent embodiments, the conjugated polyene can contain, for example,two, three, four, five, six, seven, eight, nine, or ten conjugatedcarbon-carbon double bonds.

The conjugated polyene can, in addition, include one or morecarbon-carbon triple bonds. In some embodiments, the protective agentcontains two or more carbon-carbon triple bonds conjugated with eachother. In such a case, the protective can be considered a polyyne.

In a particular embodiment, the polyene is a cyclic polyene, such as anannulene. The annulenes particularly considered herein are thosecontaining greater than six carbon atoms and/or more than threeconjugated carbon-carbon double bonds. The annulene can be aromatic ornon-aromatic. Some examples of annulenes particularly considered hereininclude cyclooctatetraene (i.e., [8]annulene or COT), [10]annulene,[12]annulene, [14]annulene, [16]annulene, and [18]annulene. The annulenemay or may not also include one or more carbon-carbon triple bonds. Theprotective agent may also be a cyclic system containing two, three,four, or more carbon-carbon triple bonds, which is herein referred to asan annulyne. The annulene or annulyne can also be functionalized withany number of hydrocarbon groups, heteroatom-functionalized formsthereof, and heteroatom groups. In particular embodiments, the annuleneor annulyne is derivatized with one, two, three, or more carboxy groups(e.g., 1,2-dicarboxycyclooctatetraene).

Typically, the conjugated polyene, annulene, or annulyne consideredherein as a protective agent is attached to an atom of another chemicalentity (e.g., fluorophore, biomolecule, or surface) either directly viaan atom of the polyene, annulene, or annulyne, or through a linker.Accordingly, any of the polyenes, annulenes, or annulynes describedabove also includes groups derived therefrom.

In another embodiment, the protective agent is a bicyclic, tricyclic, orhigher cyclic ring system containing at least two, three, or four ringnitrogen atoms. Some examples of such bicyclic molecules include1,4-diazacyclohexane, 1,4,7-triazacyclononane, and1,4,7,10-tetraazacyclododecane. Some examples of such tricyclicmolecules include 1,4-diazabicyclo[2.2.2]octane (DABCO),1,4-diazabicyclo[2.2.1]heptane, 1,5-diazabicyclo[3.2.2]nonane,1,5-diazabicyclo[3.3.2]decane, 1,5-diazabicyclo[3.3.3]undecane,1,6-diazabicyclo[4.3.0]nonane, 1,6-diazabicyclo[4.4.0]decane,1,6-diazabicyclo[4.3.3]dodecane, 1,6-diazabicyclo[4.4.3]tridecane, and1,6-diazabicyclo[4.4.4]tetradecane. The bicyclic, tricyclic, or highercyclic ring system may or may not be derivatized with one or more otherheteroatoms (e.g., oxygen, sulfur, phosphorus, and halide atoms) and/orheteroatom groups (e.g., carbonyl, ester, carboxyl, amino, amido, andthe like). The bicyclic, tricyclic, or higher cyclic ring system may ormay not also contain alkenyl or alkynyl groups.

In yet another embodiment, the protective agent is a mercaptan (i.e.,hydrocarbon group containing a —SH group). The mercaptan (i.e., thiol)can be a group on any of the hydrocarbon groups described above. Forexample, the thiol can be thiophenol, 1,4-benzenedithiol,1,3,5-benzentrithiol, a thionaphthol, or a thioanthracenol (e.g.,9-thioanthracenol). In a particular embodiment, the thiol is amercapto-substituted straight-chained alcohol, such asβ-mercaptoethanol, 3-mercaptopropanol, 4-mercaptobutanol,5-mercaptopentanol, 6-mercaptohexanol, 7-mercaptoheptanol, and8-mercaptooctanol. In another particular embodiment, the thiol is amercapto-substituted straight-chained amine, such asp-mercaptoethylamine, 3-mercaptopropylamine, 4-mercaptobutylamine,5-mercaptopentylamine, 6-mercaptohexylamine, 7-mercaptoheptylamine, and8-mercaptooctylamine. In the mercaptan compounds, the thiol group,hydroxyl group, and/or amino group can be substituted with one or morehydrocarbon groups, thereby resulting, respectively, in a thioether,ether, and secondary or tertiary amino group.

In still another embodiment, the protective agent is a phenolicderivative. Some examples of phenolic derivatives include the cresols,butylated phenols (e.g., butylated hydroxytoluene, i.e., BHT),naphthols, anthracenols (e.g., 9-anthracenol), and the like. In aparticular embodiment, the phenolic derivative is a polyphenol molecule.Some examples of polyphenol molecules include dihydroquinone, catechol,resorcinol, 1,3,5-trihydroxybenzene, gallic acid and esters thereof(e.g., n-propyl gallate and gallic acid esters of glucose or othersugar), pyrogallol, the flavonoids, flavonols, flavones, catechins,flavanones, anthocyanidins, and isoflavonoids. The phenolic derivativecan also be an etherified phenol, wherein the etherifying group can be,for example, a hydrocarbon group, particularly an alkyl group, such as amethyl, ethyl, or isopropyl group.

The nature and/or presence of the fluorescence modifier is not arequirement for fluorescence, however short the lifetime of fluorescencemay be. Rather, incorporation of the fluorescence modifier enhances thephotophysical properties of the fluorophore, including the lifetime offluorescence. The drawing shown in FIG. 20 illustrates that the modifiercan be connected to the fluorophore via a ‘linker’, [XY_(n)]_(m), andthat not only can there be more than one ‘linker’-(fluorescencemodifier) moiety per fluorophore, but there can also be more than onefluorescence modifier per linker. For example, the general structure cancontain up to five modifiers per linker and each fluorophore can containup to ten linkers. In addition, these molecules can be configured toinclude a handle such that they can be attached to reactive functionalgroups on biomolecules and/or other moieties where a fluorescent ‘tag’is required.

In the linker shown in FIG. 20, each occurrence of X can beindependently, for example, carbon, oxygen, nitrogen, sulfur, orphosphorous; each occurrence of Y can be independently, for example,hydrogen, carbon, oxygen, nitrogen, sulfur, phosphorous, an alkyl, acyl,aromatic, heteroaromatic, aryl, heteroaryl, alkyl(aryl), oralkyl(heteroaryl) group; each occurrence of n can be, independently, forexample, an integer of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or within arange therein; each occurrence of m can be, independently, for example,an integer from 0 to 20 or a subrange therein; each occurrence of x canbe independently, for example, an integer of 1, 2, 3, 4, or 5, or withina range therein; and each occurrence of y can be independently, forexample, an integer of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or within arange therein.

As used herein, a “linker” is a chemical group that connects two, three,or more chemical groups. The linker can, for example, connect afluorophore to a protective agent, and/or a fluorophore to a biomoleculeor surface, and/or a protective agent to a biomolecule or surface. Indifferent embodiments, the linker preferably contains precisely, atleast, or no more than one, two, three, four, five, six, seven, eight,nine, ten, eleven, or twelve atom-lengths between linked groups. In oneembodiment, the linker is a hydrocarbon linker, e.g., as derived fromany of the hydrocarbon groups described above by replacement of two ormore hydrogen atoms by a respective number of linking bonds. Someexamples of hydrocarbon linkers include methylene, ethylene,trimethylene, tetramethylene, pentamethylene, hexamethylene, o-, m-, andp-phenylene, and vinylene. The hydrocarbon linker may or may not alsoinclude heteroatoms. Furthermore, the heteroatoms may or may not belinking atoms. In a particular embodiment, the hydrocarbon linkercontains one, two, three, or more amino groups. Some examples ofamino-containing linkers include ethylenediamine, 1,3-diaminopropane,1,4-diaminobutane, 1,5-diaminopentane, 1,6-diaminohexane,diethylenetriamine, triethylenetetramine, and diaminobenzene linkers. Inanother particular embodiment, the hydrocarbon linker contains one, two,three, or more oxy atoms or hydroxy groups. Some examples ofoxy-containing linkers include ethylene glycol, diethylene glycol,triethylene glycol, 2-hydroxypropane, 2,3-dihydroxybutane,dihydroxybenzene, and the polyethylene glycols (i.e., PEG) linkers. Inother particular embodiments, the linkers include one, two, three, ormore carbonyl groups. Some examples of such linkers include methyldicarbonyl, ethyl-1,2-dicarbonyl, propyl-1,3-dicarbonyl,butyl-1,4-dicarbonyl, pentyl-1,5-dicarbonyl, and the like. The linkercan also be, for example, a polysulfate chain, a mono or polyphosphatechain, an oligonucleotide, oligopeptide, oligosaccharide, nucleotide,nucleoside, nucleobase, dinucleotide, trinucleotide, tetranucleotide,amino acid, dipeptide, tripeptide, tetrapeptide, saccharide,disaccharide, trisaccharide, tetrasaccharide, lipid, or fatty acid.

In particular embodiments, the linker is a rigid linker that forces atleast two linked groups to remain at fixed distances from each other orfrom another molecule. Such rigid linkers can be advantageous in keepinga protective agent group a fixed distance from a fluorophore, or twoprotective agent groups a fixed distance from each other, and/or aprotective agent. A group or fluorophore a fixed distance from abiomolecule or surface. Some examples of rigid linkers are thosecontaining aromatic or heteroaromatic rings, such as linkers thatinclude benzene, styrene, divinylbenzene, biphenyl, triphenyl, or otheraromatic rings and polycyclic ring systems. Generally, for the purposesof this application, a biomolecule (i.e., biomolecule of at least 1,000Daltons, such as a nucleic acid, protein, or polysaccharide) is notconsidered a linker.

In a first embodiment, the invention is directed to compounds in which afluorophore is attached to at least one, two, or more protective agentmoieties. The fluorophore can be attached (e.g., covalently) to theprotective agent groups by any of the linking methodologies known in theart. For example, a commercial mono-reactive fluorophore (e.g., NHS-Cy5)or bis-reactive fluorophore (e.g., bis-NHS-Cy5 or bis-maleimide-Cy5) canbe used to link to one or more protective agent molecules containingappropriate reactive groups (e.g., amino, thiol, hydroxy, aldehydic, orketonic groups). Alternatively, a protective agent molecule can bederivatized with one, two, or more reactive groups, and the reactiveprotective agent (e.g., NHS-Trolox) reacted with a fluorophorecontaining appropriate reactive groups (e.g., an amino-containingfluorophore).

In one embodiment, at least two protective agent groups are attached toa fluorophore, and the protective agent groups are within the samechemical class (e.g., two or three protective agent groups bound to thefluorophore are chromanol-derived groups or polyene groups), but may ormay not be structurally the same. In another embodiment, at least twoprotective agent groups are attached to a fluorophore, and theprotective agent groups are not within the same chemical class (e.g.,one protective agent group is a chromanol-derived or polyene group,while another protective agent group is a nitro-substituted aromaticgroup, or a bicyclic, tricyclic, or higher cyclic ring system containingat least two, three, or four ring nitrogen atoms, or a phenolicderivative). In one embodiment, one, two, or more protective agentgroups are bound directly to a fluorophore (i.e., without a linker). Inanother embodiment, one, two, or more protective agent groups are boundindirectly to a fluorophore via a linker group, such as any of thelinker groups described above. By judicious selection of where theprotective agent groups are linked on a fluorophore, the protectiveagent groups can be made to be a selected distance apart from each otherand/or from the fluorophore. For example, the protective agent groupscan be linked to the fluorophore such that they are precisely, at least,or no more than one, two, three, four, five, six, seven, eight, nine,ten, eleven, or twelve atom-lengths apart. In some embodiments, theprotective agent groups enhance or modify a photophysical property ofthe fluorophore in a manner that cannot be achieved by each of theprotective agent groups individually, i.e., when not paired. In otherembodiments, the protective agent groups, in combination, exhibit asynergistic effect in enhancing or appropriately modifying aphotophysical property of the fluorophore.

Some examples of fluorophores containing one or more reactive groups, aswell as fluorophores containing a protective agent group, are shown inFIGS. 21-24B. As shown, the fluorophore-protective agent composition canadditionally contain one or more remaining reactive groups to bind toanother molecule, e.g., another protective agent, a biomolecule, or asurface.

In an exemplary embodiment, the fluorophore-protective group compositionhas the following structure:

In an exemplary embodiment, the fluorophore-protective group compositionhas the following structure:

In an exemplary embodiment, the fluorophore-protective group compositionhas the following structure:

In an exemplary embodiment, the fluorophore-protective group compositionhas the following structure:

In an exemplary embodiment, the fluorophore-protective group compositionhas the following structure:

In an exemplary embodiment, the fluorophore-protective group compositionhas the following structure:

In an exemplary embodiment, the fluorophore-protective group compositionhas the following structure:

In an exemplary embodiment, the fluorophore-protective group compositionhas the following structure:

In an exemplary embodiment, the fluorophore-protective group compositionhas the following structure:

In an exemplary embodiment, the fluorophore-protective group compositionhas the following structure:

In another embodiment, the invention is directed to a composition inwhich a fluorophore and a protective agent molecule are not covalentlylinked directly or through a linker, but are in sufficient proximitysuch that the protective agent molecule has an effect on thephotophysical properties of the fluorophore. More particularly, thefluorophore and protective agent are close enough to mediate radiativeor collisional relaxation of the fluorophore from unwanted, unproductive(i.e., non-fluorescing) excited states or triplet dark states back toproductive fluorescing states, such as the singlet excited state, whichthen relax with concomitant fluorescence emission. For each compoundthat mediates relaxation of the fluorophore, this benefit may bestrongly distance dependent. For example, the ideal proximity of certainprotective agents, such as NBA and Trolox, is within about 1-20Angstroms. The ideal distance may vary substantially for otherprotective agent (e.g., 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50Angstroms, or a range thereof, may be ideal).

For example, a fluorophore and protective agent can each be linked to abiomolecule. The biomolecule can be, for example, a nucleic acid (e.g.,a DNA or RNA strand), protein, polysaccharide, nucleoprotein, and thelike. In particular embodiments, the fluorophore and protective agentare linked at separate specific locations of the biomolecule such that aspecified distance separates the fluorophore and protective agent. Forexample, the fluorophore and protective agent may be on adjacent unitsof the biomolecule (e.g., adjacent nucleotide, peptide, or saccharideunits), or separated by precisely, at least, or no more than one, two,three, four, five, six, seven, eight, nine, ten, eleven, twelve,thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, ortwenty units. In the case of a nucleic acid, the fluorophore andprotective agent can be independently attached to the base, phosphate,or sugar moiety. In the case of a protein, the fluorophore andprotective agent can be independently attached to, for example, theamido backbone —NH group, or particular amino acid side chains (e.g.,phenolic groups of tyrosine, or amino groups of lysine, arginine,histidine, asparagine, or glutamine, or thiol groups of cysteine, orhydroxy groups of serine or threonine, or carboxylic acid groups ofaspartate or glutamate). It is also envisaged that a biomolecule ischemically tailored to contain certain chemical groups that specificallybind to (optionally, when activated) a fluorophore and/or protectiveagent.

A fluorophore and/or protective agent can also be site-specificallyincorporated into a protein. For example, by use of orthogonalchemistries, site-specific incorporation of a fluorophore or protectiveagent can be accomplished by non-natural amino acid incorporation,post-translational modifying enzymes, or c-terminal ligation. (Referenceis made to Muir T. Annu Rev. Biochem. Vol. 72 pg. 249-89 (2003); RyuYand Schultz P G. Nat. Methods Vol. 3(4) pg 263-5 (2006); Wang L, Xie J,Deniz, A A and Schultz P G J. Org. Chem. Vol 68(1) pg 174-6 (2003);Chin, J W, Cropp T A, Anderson J C, Mukherji M, Zhang Z, Schultz P GScience Vol. 299(5615) pg. 2045-7 (2003); Warren, J. D., et al. J. Am.Chem. Soc., 2004, Vol. 126, pg. 6576-6578; Yin Y, Lin A J, Golan D E,Walsh C T. Nat Protoc. Vol. 1(1) pg. 280-5 (2006), all of which areincorporated herein by reference in their entirety).

In a particular embodiment, the biomolecule on which the fluorophoreand/or protective agent is attached is a fluorescent protein. Thefluorescent protein can be, for example, a green fluorescent protein(GFP) and its mutated allelic forms (e.g., blue, cyan, and yellowfluorescent proteins) and red fluorescent protein (RFP), and geneticvariants thereof. Another example of a fluorescent protein is mCherryand genetic variants thereof. Positions containing Tyrosine, Tryptophan,or Phenylalanine are preferred so that the introduction of non-natural,aromatic amino acid would have minimal perturbation to the system whilehaving the maximal beneficial effect. Residues must also be within 1-20Å to promote proximity effects. Specific residue to be targeted Tyr203in the active site of the protein. Selection efforts may also benecessary to screen for secondary mutations that ensure folding andstability of the protein (Reference is made to Hiem R, Cubitt A B, TsienR Nature Vol. 373(6516) pg. 663-4 (1995), which is incorporated hereinby reference in its entirety).

As another example of a fluorophore and protective agent being inproximity without being linked is an embodiment in which either thefluorophore or protective agent is incorporated into a vesicle. Thevesicle can be any vesicle known in the art capable of retainingmolecules therein. In one embodiment, the vesicle is composed of any ofthe lipid bilayer membranes (e.g., micelles, liposomes, andproteoliposomes) known in the art. As is well known in the art, theunits making up the lipid bilayer are typically amphiphilic. A commonclass of amphiphilic compounds useful for forming micelles and liposomesis the class of phospholipids; however, several other membrane units areknown. Some subclasses of the phospholipids are the phosphatidylcholinesand the phosphatidylethanolamines. In another embodiment, the vesicle iscomposed of any of the polymer membranes known in the art. Some polymermembranes include those based on polyethyleneoxides, polyesters,polyamines, polyethylenes, polystyrenes, polyurethanes, and blockcopolymers thereof. In particular embodiments, the protective agent iswithin a vesicle while the fluorophore is on the outside surface of thevesicle, or alternatively, the fluorophore is within a vesicle while theprotective agent is on the outside surface of the vesicle. By being“within” the vesicle is meant that the fluorophore or protective agentmolecule is floating within a liquid matrix within the vesicle, or thefluorophore or protective agent molecule is attached, either directly orvia a linker, to the inner wall of the vesicle. By being “outside” thevesicle is generally meant that the fluorophore or protective agentmolecule is attached, either directly or via a linker, to the outersurface of the vesicle. The bond between the fluorophore and/orprotective agent and inner or outer surface of the vesicle can becovalent or non-covalent (i.e., electrostatic, van der Waals, orhydrogen bonds). The inner and/or outer surface of the vesicle can beappropriately functionalized for this purpose by methods known in theart. The vesicle can also be either unilamellar or multilamellar. Inparticular embodiments, both the fluorophore and protective agent areeither within the vesicle or on the outer surface of the vesicle. Atleast one specific advantage of this type of configuration is for thestudy of integral- or membrane-associated biomolecule function, such asin the study of neurotransmitter or neurotransporter protein function inthe nerve synapse as related to neurotransmitter re-uptake followingsynaptic transmission. The use of vesicles also permits the study ofcell-derived membrane extract fractions that are not amenable to directsurface immobilization strategies.

In particular embodiments, the protective agent is directly attached toa lipid head group or within the lipid bilayer (e.g., by linking to asterol, such as cholesterol) so that it is in close proximity to thefluorophore, yet not covalently attached to the fluorophore. In such anembodiment, the protective agent can be attached to the external orinternal environment of the lipid vesicle. In a preferred embodiment,the vesicle surrounds the biomolecule of interest (e.g., a ribosome),thereby providing a protective and controlled environment for thebiomolecule. The foregoing embodiment can be particularly useful in theimaging of the encapsulated biomolecule. Such embodiments may utilizeone or numerous (e.g., hundreds) of protective agents that are the sameor diverse. The vesicle may also be porous so that the protective agentscan penetrate the interior in order to achieve high effectiveconcentrations, yet exchange with the interior to maintain/replenish theprotective agent concentration over time. Such encapsulation strategiescan advantageously provide for a controlled microenvironment that couldaccommodate a wide range of biomolecules diverse in both composition andsize. Alternatively, the protective agents could be attached or embeddedinto or onto a lipid bilayer that is not a vesicle or liposome. Such anapproach may include nanodiscs (e.g., Bayburt T H, Sligar S G FEBS Lett.October 16. Epub (2009) or other similar lipid bilayer architecture.Alternatively, the protective agent could be encapsulated into or onto avirus capsid such that a fluorescently labeled biomolecule of interesteither inside or outside the virus capsid benefits from the proximity ofone or more protective agents. Viral capsid proteins are robust tomutation as demonstrated by phage display technologies (e.g., Smith GPScience Vol. 228(4705) pg. 1315-7 (1985) and variants able to passscreening procedures can be amplified (Smith G P Petrenko V A Chem. Rev.Vol. 97(2) pg. 391-410 (1997). Viral capsid proteins also assemble inclose proximity so that the incorporation of non-natural amino acids(protective agents) could be densely packed.

The vesicle size is typically in the range of, e.g., about, at least, orno more than 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 5, 10,15, 20, 25, 30, 35, 40, 45, or 50 microns, or within a range therein.However, significantly smaller vesicles are possible (e.g., nanosomes),with sizes of, e.g., about, at least, or no more than 1, 2, 5, 10, 20,30, 40, 50, 60, 70, 80, or 90 nm, or within a range therein.

As another example of a fluorophore and protective agent being inproximity without being linked is an embodiment in which a protectiveagent is placed on the surface of a quantum dot nanoparticle or othernanoparticle. The protective agent can reside on the surface of thequantum dot by any mode of attachment, e.g., covalent or non-covalent(i.e., by electrostatic, van der Waals, or hydrogen bonds) modes ofattachment. In some embodiments, reactive groups suitable for theattachment of one or more protective agents is included either within oron the surface of the quantum dot, such as within the first passivationlayer present in commercially available quantum dots (see, for example,FIG. 15).

The quantum dots, either during or after their formation, are oftensurface functionalized with hydrophilic or hydrophobic groups (e.g.,surface capping groups, such as long chain thiols, carboxylates, orphosphates, or polymers). Long chain amphiphilic molecules generallyproduce an ordered capping surface on the quantum dot known in the artas a “self-assembled monolayer”. Some examples of metal-binding polymerssuitable for stabilizing or functionalizing quantum dot particlesinclude the dextrans (e.g., carboxymethyl dextran), dextroses,celluloses and their derivatives, polyethylene oxides or glycols (PEGs),albumin, and the like.

As known in the art, the surface capping groups can be functionalizedwith one or more reactive groups (e.g., amino, carboxy, epoxy, aldehyde,alkyl, halide, iodoacetamide, isocyanate, hydrazido, or semicarbazidegroups) suitable for attaching to the fluorophore, protective agent,and/or another molecule, such as a biomolecule. The protective agent maythus be incorporated in a first, second, or third passivation shell ordoped or impregnated in the core material. In some embodiments, thereactive group is capable of binding to a molecule without beingactivated in some manner, whereas in other embodiments the reactivegroup is activated in order to bind to the molecule. Alternatively, afunctional group of the fluorophore, protective agent, and/or othermolecule, such as a biomolecule, is activated or converted by a linkinggroup to a group capable of reacting with a group on the surface of thequantum dot.

A bis-reactive linking molecule can also be used to link the surface ofthe quantum dot (or other type of nanoparticle or vesicle) with afluorophore, protective agent, or other molecule, such as a biomolecule.For example, amino-amino coupling reagents can be employed to link anamino group of a passivation molecule or polymer, or vesicle membrane,with an amino group of a fluorophore, protective agent, or othermolecule. Some examples of suitable amino-amino coupling reagentsinclude diisocyanates, alkyl dihalides, dialdehydes, disuccinimidylsuberate (DSS), disuccinimidyl tartrate (DST), and disulfosuccinimidyltartrate (sulfo-DST), all of which are commercially available. Or, forexample, amino-thiol coupling agents can be employed to link a thiolgroup of a passivation molecule or polymer, or vesicle membrane, with anamino group of the fluorophore, protective agent, or other molecule.Some examples of suitable amino-thiol coupling reagents includesuccinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC), andsulfosuccinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate(sulfo-SMCC).

Other linking chemistries are possible. For example, the quantum dot,other nanoparticle, or vesicle can be linked to a fluorophore,protective agent, biomolecule, or a surface, by use of known strong andselective interactions between certain pairs of biomolecules. Forexample, in one embodiment, an amino-functionalized nanoparticle orvesicle can be reacted with an amine-reactive biotinylation reagent tofunctionalize the nanoparticle or vesicle with biotin. Some examples ofamine-reactive biotinylation reagents include the class of moleculescontaining biotin on one end and, for example, a succinimide ester,pentafluorophenyl ester, or alkyl halide group on the other end. Thebiotin group and amine-reactive group can be separated by any suitablespacer group of any length (e.g., 5-40 Å in length). Some examples ofamine-reactive biotinylation reagents are available from Pierce underthe EZ-Link® trade name, e.g., as NHS-biotin (containing a five-carbonester linkage between biotin and NHS), sulfo-NHS-biotin, NHS-LC-biotin,and sulfo-NHS-LC-Biotin, NHS-LC-LC-biotin, sulfo-NHS-LC-LC-biotin,sulfo-NHS-SS-biotin, NHS-PEO₄-biotin, PFP-biotin, TFP-PEO-biotin, andthe like, wherein “NHS” refers to a N-hydroxysuccinimide group, “LC”refers to a six-carbon amide-containing linkage inserted between the NHSgroup and biotin or between another LC group and biotin, “PEO” refers toan ethylene oxide group, wherein the associated subscript indicates thenumber of linked PEO units, “PFP” refers to a pentafluorophenyl group,“TFP” refers to a tetrafluorophenyl group, “sulfo” refers to a sulfonate(SO₃ ⁻Na⁺) group, and “SS” refers to a disulfide bond.

In another embodiment, a thiol-functionalized nanoparticle or vesiclecan be reacted with a thiol-reactive biotinylation reagent tofunctionalize the nanoparticle or vesicle with biotin. Some examples ofthiol-reactive biotinylation reagents include the class of moleculescontaining biotin on one end and, for example, a maleimido or alkylhalide group on the other end. The biotin group and thiol-reactive groupcan be separated by any suitable spacer group of any length, as above.Some examples of thiol-reactive biotinylation reagents are availablefrom Pierce under the EZ-Link® trade name, e.g., asmaleimide-PEO₂-biotin, biotin-BMCC (contains an end-maleimido group andone cyclohexyl, two amide linkages, and nine additional linking carbonatoms), PEO-iodoacetyl biotin, iodoacetyl-LC-biotin, biotin-HPDP(contains a pyridyl disulfide group), and the like.

The biotin-functionalized nanoparticle or vesicle can then be reactedwith an avidin or streptavidin-functionalized fluorophore molecule,protective agent molecule, biomolecule, or surface, such that ananoparticle or vesicle complex is produced which contains thenanoparticle or vesicle conjugated to the fluorophore and/or protectiveagent and/or biomolecule and/or surface via a biotin-avidin orbiotin-streptavidin link. In an analogous embodiment, the nanoparticleor vesicle is conjugated to avidin or streptavidin and the avidin- orstreptavidin-functionalized nanoparticle or vesicle reacted with abiotinylated fluorophore, protective agent, biomolecule, or surface,such that a nanoparticle or vesicle complex is produced which containsthe nanoparticle or vesicle conjugated to the fluorophore and/orprotective agent and/or biomolecule and/or surface via an avidin-biotinor streptavidin-biotin link. Similar embodiments are envisaged by use ofantibody pairs, or by selective metal-ligand linkages, e.g., nickel-NTAlinkage.

The fluorophore and protective agents may be linked together, eitherdirectly or via a linker, and the resulting fluorophore-protective agentcomposition attached to a molecule of interest, such as a biomolecule orbiomolecule, or a surface of a bulk solid (e.g., FIGS. 9A-9B and10A-10B). Alternatively, the fluorophore and protective agents are notlinked together, but separately attached to a biomolecule or surface ofa bulk solid. The fluorophore-protective agent composition may also beattached to a molecule, such as a biomolecule or biomolecule, eitherdirectly or via a linker. In particular embodiments, thefluorophore-protective agent composition is attached to a moleculedirectly (i.e., without an intervening conjugating group) either via afluorophore-molecule direct bond, a protective agent-molecule directbond, or via an accessible atom of a linker that connects thefluorophore and protective agent. An advantage of the method describedherein over conventional methods of the art is that, using the instantmethod, one can essentially take a commercially available bis-reactivedye and functionalize one end of it with a protective agent and thenattach the other end to a biomolecule of interest. Another advantage ofthe method used herein is in the spatial uniformity that can be impartedbetween the fluorophore and molecule of interest. This is particularlyadvantageous in view of the great reliance on fluorophores for measuringvarious properties of a molecule (ex. distance between subunits byFRET). It is known that there is an inherent error introduced into suchstudies by having some kind of spacer (i.e., one of many variablelengths) in between the fluorophore and molecule of interest. However,in a preferred embodiment of the instant invention, one cansignificantly control for that error by consistently using the samespacer for each measurement. For example, in many commercial Cy-dyes,the spacer is typically a six-carbon chain terminating in a carboxylicacid. The molecule of interest is then attached to the fluorophore atthe carboxylic acid. By leaving the linking length of the moleculeessentially unchanged, any intrinsic error will also remain unchanged.

As used herein, a “surface” refers to the surface of a bulk solidmaterial. The bulk solid material can be, for example, a particle (orbead) of an organic or inorganic polymeric composition. In oneembodiment, the surface is inorganic, e.g., silica-based. In anotherembodiment, the surface is organic, e.g., polymer-based. In a particularembodiment, the fluorophore and/or protective agent and/or biomoleculeis attached directly or via a linker to a surface using linkingchemistry well known in the art (as schematized, for example, in FIGS.2, 3, 4A-4C, 8A-8B, 9A-9B, 10A-10B, 11A-11D, 12A-12C, 13A-13B, 14, 15,and 17A-17B). In some embodiments, a fluorophore and protective agentare each attached to the surface proximally to each other. In otherembodiments, a fluorophore-conjugated biomolecule and one, two, or moreprotective agents are each attached to a surface proximally to eachother (FIGS. 13A, 13B-16). In other embodiments, a protectiveagent-conjugated biomolecule and one, two, or more fluorophore moleculesare each attached to a surface proximally to each other (FIGS. 13A,13B-16). In other embodiments, a biomolecule conjugated to one or morefluorophores and one or more protective agents is attached to a surfaceproximally to one or more fluorophores and/or protective agents and/orbiomolecules conjugated to a fluorophore and/or protective agent. (FIGS.8A-8B, upper right and lower left panels). The particular embodimentsshown in FIGS. 8A-8B and 9A-9B provide means of establishing proximitybetween the fluorescent species and the protective agent throughnon-covalent attachment strategies. Numerous other variations andstrategies related to those shown in FIGS. 8A-8B and 9A-9B are possible.

The fluorophore compositions described herein can be used in any methodor technology in which fluorophores are used. In a particularembodiment, the fluorophore compositions described herein are applied tofluorescence-based assay methods, such as PCR and ELISA assay methods.In more particular embodiments, the fluorophore compositions describedherein are applied to FRET methods, and more particularly, smFRETmethods. These methods are well known in the art. Particular referenceis made to R. Dave, et al., Biophysical Journal, vol. 96, March 2009,pp. 2371-2381; Stryer L. Annu Rev. Biochem. Vol. 47 pg. 819-46 (1978);Forster T. (Ann Physik (1959); Roy R. Hohng S, Ha T. Nature Methods Vol.5(6) pg. 507-516 (2008). Weiss SR Science Col. 283(5408) pg. 1676-83(1999), all of which are incorporated herein in its entirety.

A significant advantage of the compositions described herein is that theposition of one or more protective agents can be adjusted and fixedrelative to one or more fluorophores. By this feature, one or morephotophysical characteristics of the fluorophore can be suitablyadjusted, optimized, or tuned to suit a particular application. Somephotophysical characteristics include, for example, fluorescencelifetime, absorption and emission wavelength and extinction, stochasticblinking events, blinking frequency, and photobleaching characteristics.The characteristics being adjusted or optimized can be characteristicsparticularly relevant to non-assay applications, such as for photonicand photoswitching devices, including organic light emitting diodes(OLEDs). Significantly, the tunability feature of the instantfluorophore-protective agent compositions allows for altering (i.e.,increasing or decreasing) the blinking rate of the fluorophore. Forexample, in certain applications, a faster blinking frequency isdesired, while in other applications, a slower blinking frequency isdesired, relative to the original blinking frequency (FIG. 11) (i.e.,blinking frequency of the fluorophore when not in proximity to aprotective agent). In other embodiments, the lifetimes of fluorescentand dark states can be tuned (as suggested by FIG. 1) by decreasing theeffective rate of transition into or out of the triplet dark state.

In another embodiment, the invention is directed to applying any of thefluorophore compositions described above to methods for detecting acellular process in a living cellular or multicellular organism. Such invivo methods often include administering to the organism an effectiveamount of the fluorophore composition, and detecting the fluorophore inthe organism. The organism being studied can be, for example, a mammal,a cell from a cell line (e.g., CHO cells or stem cells), a microbe(e.g., a bacterium or protozoan), or a mammalian or non-mammalian eggcell. Typically, the fluorophore composition to be administeredpossesses a portion (i.e., chemical group) that specifically andselectively targets a biological site or particular biomolecule in themammal. Therefore, the fluorophore composition used in this mannerfunctions as a targeting probe. These fluorophore compositions can alsocircumnavigate cell membrane permeability issues and the potentialtoxicity of protective agents in solution to a living cell. Furthermore,in some embodiments, the protective agent itself can function as a cellpermeation enhancer. The specific application of this approach relatesto the site-specific labeling of one or more target molecules in thecell by adding the fluorescent species to the cell medium or animalcirculation. In both cases, crossing the cell membrane can be a limitedaspect of the approach.

Examples have been set forth below for the purpose of illustration andto describe the best mode of the invention at the present time. However,the scope of this invention is not to be in any way limited by theexamples set forth herein.

EXAMPLES

Experimental Materials and Methods

Preparation of Dye-Labeled/Protective Agent Linked Oligonucleotides

Cy3, 5 or 5.5 Bis NHS-Ester was first reacted with an equimolar amountof amine modified protective agent in 50 mM potassium borate pH8 for 10minutes at 37 degrees. After quenching with 2 fold excess of potassiumhydroxide, monoreacted dye was purified from unreacted and di-reacteddyes using a semipreparative HPLC C18 column in 10 mM triethylammoniumacetate in a gradient from 25-100% acetonitrile. After evaporation ofacetonitrile, the product was concentrated and buffer exchanged over asep-pak C18 column and eluted in methanol before final lyophilization.Reactive functional groups are regenerated as shown in FIGS. 6A-6D.

Two complimentary 12 or 21-nucleotide DNAs were chemically synthesizedwith one strand possessing a 5′-C₆-amino linker for dye linkage alongwith an additional 3′-biotin moiety attached via a 22 atom spacer(5′-/5AmMC6/GTC ATG GTC ATG/3BioTEG/-3′ or 5′-/SAmM C6/CAT GAC CAT GACCAT GAC CAG/3BioTEG/-3′ IDT, Coralville, Iowa) and the complimentarystrand being either 5′-/5 AmM C6/CAT GAC CAT GAC-3′ or 5′-CTG GTC ATGGTC ATG GTC ATG-3′ with a C₆-amino linker incorporated at selectpositions within the sequence. Each DNA strand was individually labeledwith either a commercially-available, N-hydroxysuccinimide (NHS) esteractivated dye molecule Cy3, Cy5 or Cy5.5; GE Healthcare, Piscataway,N.J.), newly generated protective agent-linked compounds, or NHSmodified protective agents through the following procedure:

Lyophilized DNAs were resuspended in distilled, deionized water andadjusted to 50 uM in 50 mM potassium borate buffer, pH 8.1, 200 mM KCl.Labeling was achieved by adding a 10-fold molar excess of NHS-reactivedye or NHS modified protective agents resuspended in Dimethyl sulfoxide(DMSO) in a 10 ul reaction at 37° C. for 10 minutes. All labelingreactions were quenched with 0.2 ul of 1M Tris pH 7.5 at 25° C. for 2minutes. Labeled strands were hybridized by mixing the 2 in equimolarratios, briefly heating to 75° C. for 12-mers and 95° C. for 21-mersfollowed by passive cooling to room temperature. Unbound dye was removedusing 200 ul DEAE sepharose resin equilibrated with 10 mM Tris AcetatepH 7.5, 200 mM ammonium chloride. Hybrids were diluted in column bufferfor binding, then after extensive washing to liberate free dye, the DNAwas eluted with 10 mM Tris Acetate pH 7.5, 1M ammonium chloride. Labeledfractions were pooled by visualization using a Kodak Gel Logic 440imaging station (˜200 ul total), diluted to 1 ml with Buffer A (1.7MAmmonium Sulfate, 10 mM Ammonium Acetate pH 5.85) and applied over anFPLC phenyl 5PW column (Tosoh Bioscience) using a 60 min gradient frombuffer A to B (10% Methanol, 10 mM Ammonium Acetate pH 5.85). The peakof interest was collected and diluted as needed for single-moleculeimaging experiments.

Single-Molecule Imaging and Experimental Conditions

All experiments were performed using a laboratory built, prism-basedTIRF apparatus as previously described at specified illuminationintensities in Tris-polymix buffer (50 mM Tris-OAc pH 7.5, 100 mM KCl, 5mM NH₄OAc, 0.5 mM Ca(OAc)₂, 15 mM Mg(OAc)₂, 0.1 mM EDTA, 50 mMβ-Mercaptoethanol, 5 mM putrescine, and 1 mM spermidine) containing 2unit/μl Glucose Oxidase (Sigma-Aldrich, St. Louis, Mo.), 20 units/μlcatalase (Sigma-Aldrich, St. Louis, Mo.) and 0.1% v/v glucose unlessstated otherwise (Munro J B Altman R B, O'Connor N, Blanchard S C, Mol.Cell Vol. 25 pg. 505-517 (2007). Biotinylated DNA molecules wereimmobilized via a biotin-streptavidin interaction within microfluidicchannels constructed on quartz slides (Blanchard S C, Kim H D, GonzalezR L, Puglisi J D, Chu S PNAS Vol. 101(35) pg. 12893-8 (2004).Fluorescence from surface-immobilized molecules, illuminated via theevanescent wave generated by total internal reflection of 532 nm (LaserQuantum, Cheshire, U.K.) and/or 635 nm (Coherent, Auburn, Calif.) lasersources, was collected using a 1.2 NA 60× water-immersion objective(Nikon, Melville, N.Y.) and imaged onto a Cascade 128B CCD (RoperScientific, Tucson, Ariz.). Data were acquired using Metamorph software(Universal Imaging Corporation, Downingtown, Pa.) collecting at a framerate of 25/sec (40 millisecond time resolution). For direct 635 nmillumination experiments, data were collected at a frame rate of 10/sec(100 millisecond time resolution).

Kinetic Analysis of smFRET Time Traces

The photophysical properties of dyes were investigated by extractingsingle-molecule time traces from the acquired CCD images using in-housedesigned software in Matlab (Natick, Mass.) as previously describedMunro J B Altman R B, O'Connor N, Blanchard S C, Mol. Cell Vol. 25 pg.505-517 (2007). FRET efficiencies (E_(FRET)) for each trace werecalculated according to the equation:

${E_{FRET} = \frac{I_{acceptor}}{I_{donor} + I_{acceptor}}};$where I_(acceptor) and I_(donor) correspond to Cy5 and Cy3 fluorescenceintensity, respectively, after correcting for background intensity andcross talk between the donor and acceptor fluorescence signals. In theanalysis of smFRET data, only those molecules yielding both donor andacceptor fluorescence were considered for analysis. In fluorescence andFRET imaging experiments, molecules yielding a signal-to-noise (S/N)ratio less than 6:1 were excluded from analysis. Unless otherwisestated, S/N is defined as the total fluorescence intensity(donor+acceptor)/standard deviation of background fluorescence afterphotobleaching. The kinetic parameters of blinking and photobleachingwere extracted from smFRET trajectories by idealizing fluorescence andFRET data to specific kinetic models using a segmental k-means algorithmimplemented in QuB as previously reported (Munro J B Altman RB, O'ConnorN, Blanchard S C, Mol. Cell Vol. 25 pg. 505-517 (2007); Dave, R et al.,Biophysical Journal, vol. 96, March 2009, pp. 2371-2381 Single-moleculedata obtained from individual DNA molecules were fit to a two-statekinetic model yielding a series of dwells in the non-zero andzero-fluorescence/FRET states. Because under all smFRET conditionstested the vast majority (>85%) of FRET trajectories were limited byacceptor photobleaching, only dwells prior to the last acceptorfluorophore dark state were examined. For direct and FRET-basedillumination experiments, individual dwells in non-zero and zerofluorescent/FRET states were used to estimate t_(on), the lifetime ofthe fluorescence state. From these parameters, the lifetimes that eachsingle-molecule was observed in the fluorescent state was estimated byfitting each distribution to exponential decay processes using theOrigin software package.

FIGS. 8A-8B presents experimental data showing two examples of linking aprotective group to Cy3. Under direct excitation of Cy3, individualdwells in the fluorescent state were used to estimate time_(on)(t_(on)), the lifetimes in the fluorescent state. This distribution wasfit to an exponential decay process to demonstrate changes relative toCy3 without a PA. Cy3 fluorescence (t_(on)) is only marginally affectedby its direct linkage to NBA, while improved approximately 2.5-fold whendirectly linked to Trolox. Cy3- and Cy3-PA fluorescence behaviors wereinterrogated in the context of its linkage to DNA as schematized usingthe single-molecule TIR scheme show in FIG. 2. The specific dyemolecules examined are shown. These data demonstrate that distinctprotective agents operate either through distinct mechanisms or todifferent extents on the Cy3 fluorophore.

FIGS. 9A-9B presents experimental data taken as described in FIGS. 8A-8Bshowing the lifetime of Cy5 fluorescence (t_(on)) is greatly improved(>10-fold) by its direct linkage to either NBA or Trolox. Cy5- andCy5-PA fluorescence behaviors were interrogated in the context of itslinkage to DNA as schematized using the single-molecule TIR scheme shownin FIG. 2. The specific dye molecules examined are shown. These datademonstrate that the same protective agents examined for the Cy3fluorophore operate either through distinct mechanisms or to differentextents on the Cy5 fluorophore.

FIGS. 10A-10B presents experimental data taken as described in FIGS.8A-8B showing the lifetime of Cy5.5 fluorescence (t_(on)) is greatlyimproved (>20-fold) by its direct linkage to either NBA or Trolox. Cy5.5and Cy5.5-PA fluorescence behaviors were interrogated in the context ofits linkage to DNA as schematized using the single-molecule TIR schemeshown in FIG. 2. The specific dye molecules examined are shown. In yetanother example that distinct protective agents operate either throughdistinct mechanisms or to different extents on fluorophore species,Trolox and NBA are shown to improve the photophysical properties of theCy5.5 fluorophore more substantially than both Cy3 and Cy5.

FIGS. 11A-11D presents experimental data demonstrating improvements inthe informative FRET lifetime for the Cy3 and Cy5 pair. (Left panels)specific samples interrogated in which no protective agent is present,NBA is linked to Cy5, Trolox is linked Cy5 or a sample where distinctprotective agents are linked to each fluorophore: NBA is linked to Cy5and Trolox is linked Cy3. (Middle panels) Experimental single-moleculeFRET data showing examples of Cy3 and Cy5 fluorescence. Experimentaldata showing the population FRET behaviors observed where each of theconstructs containing fluorophore-PA conjugates has an extended FRETduration. In the sample where each fluorophore of the FRET pair islinked to a protective agent (bottom panels), the total lifetime of FRETis greatly improved over samples containing no protective agents(>20-fold) and significantly improved over samples where only theCy5-fluorophore is linked to a protective agent (>2-fold) (note the timeaxes on the bottom panels represent a much longer imaging period thanthe others shown). By increasing the FRET duration and minimizing darkstates, the information content of the experiment is substantiallyimproved. By extending the FRET duration, more robust structuralinformation can be obtained (as required for “Molecular EKG” imaging(Blanchard S C Curr Opin Struct. Biol Vol. 19(1) pg. 103-9 (2009))and/or greater confidence in the localization of single-fluorophoreswhen tracking blinking kinetics (as for super-resolution imaging).

FIGS. 12A-12C presents experimental data demonstrating another exampleof the improvement in FRET lifetimes between a different FRET pair. Inthis case Cy3 and Cy5.5 are interrogated as described in FIGS. 11A-11D.Population histograms (right panels) reveal an increase in the lifetimeof fretting molecules when either of two protective agents are linked tothe Cy5.5 acceptor dye. Significantly, these data demonstrate anotherexample showing that protective agents linked to the acceptorfluorophore, Cy5.5 greatly extend the total period of FRET observationcompared to the case when no protective agents are present (>5-fold).

FIGS. 13A-13B presents experimental data showing the distance dependenceof protective agent affects on fluorophore performance using Cy5-labeledDNA as a specific example. Protective agents NBA (left panel) or Trolox(right panel) were linked to 21-mer DNA oligonucleotides either directlythrough an NHS activating group on Cy5, or proximal to the fluorophorethrough a modified amine at the C6 position of internally designatedbases. Under direct excitation of Cy5, individual dwells in thefluorescent state were used to estimate t_(on), the lifetimes in thefluorescent state. This distribution was fit to a double exponentialdecay process and the time constants were divided by that of a no-PAcontrol to obtain the fold protection for each individual distance.These data demonstrate that the PA need not be directly attached to thefluorophore species in order to mediate control over the fluorophorephotophysics.

In particular, FIGS. 13A-13B includes drawings and charts depicting howproximity of the protective agent to the fluorophore mediates protectionin a distance-dependent fashion. Exemplary proximity relationships forCy5-NBA and Cy5-Trolox are shown, where either protective agent wasmoved away from the fluorophore by linking it through a DNAoligonucleotide spacer. Triplet state quenchers NBA (left panel) orTrolox (right panel) were linked to 21-mer DNA oligonucleotides eitherdirectly through an NHS activating group on Cy5, or proximal to thefluorophore through a modified amine at the C6 position of internallydesignated bases. Under direct excitation of Cy5, individual dwells inthe fluorescent state were used to estimate “time on” (i.e., t_(on)),the lifetime in the fluorescent state. This distribution was fit to adouble exponential decay process and the time constants were divided bythat of a no-TSQ control to obtain the fold protection for eachindividual distance.

FIG. 14 presents drawings depicting some exemplary specific,non-covalent geometries and means for establishing close proximitybetween fluorescent molecules and one or more protective agents usingCy5-labeled DNA as a specific example (Upper left panel) proximity ofone or more protective agents to a fluorescing species by its attachmentto a nearby secondary molecule or chemical post (purple block). (Upperright panel) proximity of one or more protective agents to a fluorescingspecies by its attachment to a secondary molecule that non-covalentlyassociates with a lipid structure encapsulating the fluorescing species.(Lower left panel) proximity of one or more protective agents to afluorescing species by its attachment to a secondary molecule thatnon-covalently associates with the fluorescing species. (Lower rightpanel) proximity of one or more protective agents to amembrane-encapsulated fluorescing species achieved through theirattachment to, or embedding within, the inner or outer leaflet of alipid vesicle, liposome or nanosome.

FIG. 15 presents drawings depicting some exemplary strategies forachieving proximity of one or more protective agents to a fluorescingspecies (Upper left panel) protective agents either chemically linked tonatural amino acid side chains located on the surface of a fluorescentprotein or by way of non-natural amino acid incorporation at the levelof protein synthesis. (Upper right panel) one or more protective agentslinked to the outer shell layer(s) of a quantum dot by way ofimpregnation or covalent attachment. (Lower left panel) proximity of oneor more protective agents to a membrane-bound fluorescing speciesachieved through the attachment to, or embedded within, the inner orouter leaflet of a lipid vesicle, liposome or nanosome. (Lower rightpanel) proximity of one or more protective agents to a fluorescencespecies via embedding or impregnating the solid support to which thefluorescent species is located proximally.

FIG. 16 presents drawings depicting some exemplary strategies forachieving proximity of more than one protective agent to a fluorescingspecies that is attached to a target or substrate of interest as well asspecific examples of Cy5-PA linkages.

FIGS. 17A-17B presents experimental data demonstrating the utility ofachieving proximity of more than one protective agent to a fluorescingspecies using Cy5-labeled DNA as an example. As shown in FIGS. 9A-9B,the Cy5-Trolox conjugate (orange) is greatly improved in lifetime overthe no-PA Cy5 control (>10-fold). By adding a second protective agent(either NBA and Trolox) in proximity (within 30 Å) of the Cy5-Troloxfluorophore, an additional improvement in fluorophore lifetime (t_(on))is observed. In the case of a Trolox (purple), the additionalimprovement is >1.3-fold. In the case of NBA (green) the additionalimprovement is >2-fold. Thus, the total improvement achievedwas >20-fold over the parent compound. Drawings depicting otherexemplary strategies for achieving proximity of more than one protectiveagent to a fluorescing species.

FIG. 18 shows a schematic representation of the tuning of fluorescencebehavior. The relevant parameters are the lifetimes in fluorescingstates (Time on, t_(on)) and non-fluorescing states (Time off, t_(off))and the fluorescence intensities. The present invention describes howone can change the intrinsic fluorescent properties of a fluorescingspecies by controlling fluorophore proximity to one or more protectiveagents. Such strategies can be used to achieve long-lasting fluorescingstates and shorter dark states or shorter-lived fluorescent and darkstates depending on the specific PAs employed and their proximity to thefluorescent species. Similar strategies may also be used to control thequantum yield (brightness) of a fluorophore under a given illuminationintensity.

The lifetimes in either state are determined by stochastic processesthat depend on intrinsic and extrinsic factors. The characteristicswitching behaviors of fluorescing species may be unwanted in the caseof single-molecule FRET imaging of a biological process, wherenon-fluorescing states may be uninformative. Alternatively, thecharacteristic switching behaviors of fluorescing species may beleveraged for determining that only one fluorophore is under observation(identified by single-step photobleaching), for determining the locationof a single fluorophore through FIONA and super-resolution imagingtechniques (Yildez A et al. Science Vol 300(5628) pg. 2061-5 (2003);Churchman L S et al. Biophys J Vol. 90(2) pg. 668-71 (2006); and Bates Met al. Science Vol. 317(5845) pg. 1749-53 (2007)), or for other imagingor computational purposes. In cases where switching behaviors are notwanted such as “molecular EKG” approaches (Blanchard S C Curr OpinStruct. Biol Vol. 19(1) pg. 103-9 (2009)), t_(on) can be maximized byusing one or more PAs and PA linkage strategies that extend the lifetimeof fluorescence, while simultaneously t_(off) should be minimized(center panel). In cases where switching behaviors are desired such asFIONA and super-resolution imaging, rapid switching may be beneficial tothe experiment in which case one or more PAs and PA linkage strategiesshould be employed to control the frequency of switching behavior andthe total time that switching behaviors are observed. In the case ofsuper-resolution imaging, fast switching for extended periods cangreatly reduce the total time needed to perform the experiment and toobtain high-resolution data.

FIG. 19 presents experimental data showing that under conditions similarto those found in the cell (buffers saturated (1-2 mM) with molecularoxygen (O₂)) fluorophore-PA proximity extends the lifetime offluorescence (t_(on)). (Upper panel) Specific, exemplary fluorophore-PAconjugates under direct excitation (namely Cy5-trolox and Cy5-NBA)(Lower panel) Quantum dots bathed in a solution of 1 mM Trolox, COT andNBA. In each case the distribution of fluorescence lifetimes (ton) werefit to an exponential process.

Reaction Procedures for the Synthesis of Dye-Protective AgentConstructs:

Analytical Equipment:

HPLC: All separations involved a mobile phase of 10 mM triethylammoniumacetate in water, pH 8.1 (solvent A)/acetonitrile (solvent B).Preparative, semi-preparative, and analytical HPLC separations wereperformed using a Varian PrepStar solvent delivery system equipped witha Varian 335 ProStar PDA detector and Varian Pursuit XRS C-18 10μcolumn, 250 mm in length and 21.2 mm in diameter (semiprep), or PursuitXRS C-18 3μ column 250 mm in length and 4.6 mm in diameter (analytical).Separations were performed at flow rates of 20 mL/min for semiprep and 1mL/min for analytical with PDA monitoring from 215 to 900 nm. Unlessotherwise mentioned, all separations were run with the followinggradient: 0-2.5 min (25% B); 2.5-12.5 min (25-95% B); 12.5-15 min (95%).All separations were complete after 15 min. All retention times (rt) arefor the analytical column following purification.

ESMS: Electrospray mass spectroscopy analysis was obtained on a WatersSQ mass spectrometer.

Exemplary reaction of a bis-(NHS) reactive cyanine dye with a protectiveagent.

To a solution of water (130 μL) and 500 mM aqueous potassium borate (20μL) was added bis-NHS-Cy5 (1 mg, 1.1 μmol) in DMSO (50 μL), immediatelyfollowed by NBA-NH₂ (2.5 μL of a 500 mM solution in DMSO). The resultingreaction was vortexed and then sat at 37° C. for 10 min at which pointit was quenched by the addition of 250 mM aqueous potassium hydroxide(10 μL). The entire reaction mixture was then injected directly onto theHPLC for purification (rt)=6.15 min. The desired compound (NBA-Cy5-COOH)was obtained as a dark blue powder following lyophilization.

Exemplary activation of a protective agent-dye-acid as an NHS ester.

To a solution of NBA-Cy5-COOH (10 nmol) in dry DMF (10 μL) was addedSbTMU (90 nmol) in dry DMF (25 μL) followed by diisopropylethylamine(DIEA, 80 nmol). The reaction was vortexed and then sat at ambienttemperature for 30 min at which point ethyl acetate (1.2 mL) was addedto affect an immediate precipitation. The mixture was centrifuged andthe supernatant was decanted. The remaining pellet was triturated withethyl acetate (×2), each time removing the supernatant aftercentrifugation. The crude product was a single peak by HPLC (rt=9.96min) and was used without further purification.

Exemplary activation of a protective agent-dye-acid as a maleimide.

To a solution of N-(2-aminoethyl)maleimide) trifluoroacetate salt (500nmol) in dry DMF (50 μL) was added a solution of NBA-Cy5-NHS (5 nmol) indry DMF (5 μL) followed by a solution of DIEA (250 nmol) in dry DMF (50μL). The reaction was vortexed and then sat at ambient temperature for30 min at which point ethyl acetate (1.2 mL) was added to affect animmediate precipitation. The mixture was centrifuged and the supernatantwas decanted. The remaining pellet was triturated with ethyl acetate(×2), each time removing the supernatant after centrifugation. The crudeproduct was a single peak by HPLC, (rt=9.05 min) and was used withoutfurther purification. HRMS: Calcd. for C₅₂H₆₃N₇O₁₂S₂ [M+Na]⁺ 1064.3874.Found 1064.3849.

Similar protocols were followed to provide the Trolox derivatives:

Trolox-Cy5-COOH was obtained as a dark blue powder followinglyophilization, rt=8.54 min.

Trolox-Cy5-NHS was obtained as a dark blue powder followinglyophilization, rt=9.13 min. HRMS: Calcd. for C₅₇H₇₁N₅O₁₄S₂ [M+Na]⁺1136.4337. Found 1136.4335.

Trolox-Cy5-Mal was obtained as a dark blue powder followinglyophilization, rt=9.30 min.

What is claimed is:
 1. A composition comprising a fluorophore havingattached thereto at least one triplet state quencher, wherein the atleast one triplet state quencher is attached covalently to thefluorophore either directly or via a linker, wherein said triplet statequencher has the ability to alter the singlet-triplet occupancydistribution of excited and relaxing electrons of a fluorophore, and thetriplet state quencher is selected from: (i) chromanol-derived groupshaving the formula:

wherein, in Formula (9), R₁, R₂, R₃, R₄ and R₅ are independentlyselected from hydrogen atom and hydrocarbon groups, wherein one of R₁,R₂, R₃, R₄ and R₅ is a bond connecting the chromanol-derived group tosaid fluorophore; (ii) nitro-substituted aromatic groups having theformula:

wherein, in Formula (12), R₁, R₂, R₃, R₄, R₅, R₆, R₇, and R₈ areindependently selected from hydrogen atom and hydrocarbon groups,wherein at least one of R₄, R₅, R₆, R₇, and R₈ is replaced with the —NO₂group, and wherein one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, and R₈ is a bondconnecting the nitro-substituted aromatic group to said fluorophore; and(iii) cyclic polyene groups containing more than three conjugatedcarbon-carbon double bonds.
 2. The composition of claim 1, wherein atleast two of said triplet state quenchers are attached to saidfluorophore.
 3. The composition of claim 1, wherein said linker has asize of less than 1,000 Daltons.
 4. The composition of claim 1, whereinsaid composition is conjugated to a biomolecule or a surface of a bulksolid by either a direct bond or by a conjugating group connecting saidbiomolecule or surface and at least one of the fluorophore or said atleast one triplet state quencher.
 5. The composition of claim 1, whereinsaid fluorophore exhibits an emission wavelength greater than 594 nm. 6.The composition of claim 1, wherein said triplet state quencher isselected from cyclic polyene groups containing more than threeconjugated carbon-carbon double bonds.
 7. The composition of claim 1,wherein R₁, R₂, and R₃ under Formula (9) are independently selected fromhydrogen, methyl, ethyl, n-propyl, and isopropyl groups; R₄ underFormula (9) is independently selected from hydrogen, methyl, ethyl,n-propyl, isopropyl, and carboxylic acid groups; and R₅ under Formula(9) is independently selected from hydrogen, methyl, ethyl, n-propyl,isopropyl, and long chain hydrocarbon groups having at least four carbonatoms.
 8. The composition of claim 1, wherein R₁, R₂, R₃, R₄, R₅, R₆,R₇, and R₈ under Formula (12) are independently selected from hydrogen,methyl, ethyl, n-propyl, and isopropyl groups.